Chromosome-specific staining to detect genetic rearrangements

ABSTRACT

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML) and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

BACKGROUND OF THE INVENTION

[0001] Chromosome abnormalities are associated with genetic disorders,degenerative diseases, and exposure to agents known to causedegenerative diseases, particularly cancer, German, “Studying HumanChromosomes Today,” American Scientist, Vol. 58, pgs. 182-201 (1970);Yunis, “The Chromosomal Basis of Human Neoplasia,” Science, Vol. 221,pgs. 227-236 (1983); and German, “Clinical Implication of ChromosomeBreakage,” in Genetic Damage in Man Caused by Environmental Agents,Berg, Ed., pgs. 65-86 (Academic Press, New York, 1979). Chromosomalabnormalities can be of several types, including: extra or missingindividual chromosomes, extra or missing portions of a chromosome(segmental duplications or deletions), breaks, rings and chromosomalrearrangements, among others. Chromosomal or genetic rearrangementsinclude translocations (transfer of a piece from one chromosome ontoanother chromosome), dicentrics (chromosomes with two centromeres),inversions (reversal in polarity of a chromosomal segment), insertions,amplifications, and deletions.

[0002] Detectable chromosomal abnormalities occur with a frequency ofone in every 250 human births. Abnormalities that involve deletions oradditions of chromosomal material alter the gene balance of an organismand generally lead to fetal death or to serious mental and physicaldefects. Down syndrome can be caused by having three copies ofchromosome 21 instead of the normal 2. This syndrome is an example of acondition caused by abnormal chromosome number, or aneuploidy. Downsyndrome can also be caused by a segmental duplication of a subregion onchromosome 21 (such as, 21q22), which can be present on chromosome 21 oron another chromosome. Edward syndrome (18+), Patau syndrome (13+),Turner syndrome (XO) and Kleinfelter syndrome (XXY) are among the mostcommon numerical aberrations. [Epstein, The Consequences of ChromosomeImbalance: Principles, Mechanisms and Models (Cambridge Univ. Press1986); Jacobs, Am. T. Epidemiol, 105:180 (1977); and Lubs et al.,Science, 169:495 (1970).]

[0003] Retinoblastoma (del 13q14), Prader-Willi syndrome (del15q11-q13), Wilm's tumor (del 11p13) and Cri-du-chat syndrome (del 5p)are examples of important disease linked structural aberrations. [Noraand Fraser, Medical Genetics: Principles and Practice, (Lea and Febiger1989).]

[0004] Measures of the frequency of structurally aberrant chromosomes,for example, dicentric chromosomes, caused by clastogenic agents, suchas, ionizing radiation or chemical mutagens, are widely used asquantitative indicators of genetic damage caused by such agents,Biochemical Indicators of Radiation Injury in Man (International AtomicEnergy Agency, Vienna, 1971); and Berg, Ed. Genetic Damage in Man Causedby Environmental Agents (Academic Press, New York, 1979). A host ofpotentially carcinogenic and teratogenic chemicals are widelydistributed in the environment because of industrial and agriculturalactivity. These chemicals include pesticides, and a range of industrialwastes and by-products, such as halogenated hydrocarbons, vinylchloride, benzene, arsenic, and the like, Kraybill et al., Eds.,Environmental Cancer (Hemisphere Publishing Corporation, New York,1977). Sensitive measures of chromosomal breaks and other abnormalitiescould form the basis of improved dosimetric and risk assessmentmethodologies for evaluating the consequences of exposure to suchoccupational and environmental agents.

[0005] Current procedures for genetic screening and bio- logicaldosimetry involve the analysis of karyotypes. A karyotype is theparticular chromosome complement of an individual or of a related groupof individuals, as defined both by the number and morphology of thechromosomes usually in mitotic metaphase. It includes such things astotal chromosome number, copy number of individual chromosome types(e.g., the number of copies of chromosome X), and chromosomalmorphology, e.g., as measured by length, centromeric index,connectedness, or the like. Chromosomal abnormalities can be detected byexamination of karyotypes. Karyotypes are conventionally determined bystaining an organism's metaphase, or otherwise condensed (for example,by premature chromosome condensation) chromosomes. Condensed chromosomesare used because, until recently, it has not been possible to visualizeinterphase chromosomes due to their dispersed condition and the lack ofvisible boundaries between them in the cell nucleus.

[0006] A number of cytological techniques based upon chemical stainshave been developed which produce longitudinal patterns on condensedchromosomes, generally referred to as bands. The banding pattern of eachchromosome within an organism usually permits unambiguous identificationof each chromosome type, Latt, “Optical Studies of Metaphase ChromosomeOrganization,” Annual Review of Biophysics and Bioengineering, Vol. 5,pgs. 1-37 (1976). Accurate detection of some important chromosomalabnormalities, such as translocations and inversions, has required suchbanding analysis.

[0007] Unfortunately, such conventional banding analysis requires cellculturing and preparation of high quality metaphase spreads, which istime consuming and labor intensive, and frequently difficult orimpossible. For example, cells from many tumor types are difficult toculture, and it is not clear that the cultured cells are representativeof the original tumor cell population. Fetal cells capable of beingcultured need to be obtained by invasive means and need to be culturedfor several weeks to obtain enough metaphase cells for analysis. In manycases, the banding patterns on the abnormal chromosomes do not permitunambiguous identification of the portions of the normal chromosomesthat make them up. Such identification may be important to indicate thelocation of important genes involved in the abnormality. Further, thesensitivity and resolving power of current methods of karyotyping arelimited by the fact that multiple chromosomes or chromosomal regionshave highly similar staining characteristics, and that abnormalities(such as deletions) which involve only a fraction of a band are notdetectable. Therefore, such methods are substantially limited for thediagnosis and detailed analysis of contiguous gene syndromes, such aspartial trisomy, Prader-Willi syndrome [Emanuel, Am. J. Hum. Genet.,43:575 (1988); Schmickel, J. Pediatr. 109:231 (1986)] and retinoblastoma[Sparkes, Biochem. Biophys. Acta. 780:95 (1985)].

[0008] Thus, conventional banding analysis has several importantlimitations, which include the following. 1) It is labor intensive, timeconsuming, and requires a highly trained analyst. 2) It can be appliedonly to condensed chromosomes. 3) It does not allow for the detection ofstructural aberrations involving less than 3-15 megabases (Mb),depending upon the nature of the aberration and the resolution of thebanding technique [Landegren et al., Science, 242:229 (1988)]. Thisinvention provides for probe compositions and methods to overcome suchlimitations of conventional banding analysis.

[0009] The chemical staining procedures of the prior art providepatterns over a genome for reasons not well understood and which cannotbe modified as required for use in different applications. Such chemicalstaining patterns were used to map the binding site of probes. However,only occasionally, and with great effort, was in situ hybridization usedto obtain some information about the position of a lesion, for example,a breakpoint relative to a particular DNA sequence. The presentinvention overcomes the inflexibility of chemical staining in that itstains a genome in a pattern based upon nucleic acid sequence; thereforethe pattern can be altered as required by changing the nucleic acidsequence of the probe. The probe-produced staining patterns of thisinvention provide reliable fundamental landmarks which are useful incytogenetic analysis.

[0010] Automated detection of structural abnormalities of chromosomeswith image analysis of chemically stained bands would require thedevelopment of a system that can detect and interpret the bandingpatterns produced on metaphase chromosomes by conventional techniques.It has proven to be very difficult to identify reliably by automatedmeans normal chromosomes that have been chemically stained; it is muchmore difficult to differentiate abnormal chromosomes having structuralabnormalities, such as, translocations. Effective automated detection oftranslocations in conventionally banded chromosomes has not beenaccomplished after over a decade of intensive work. The probe-producedbanding patterns of this invention are suitable for such automateddetection and analysis.

[0011] In recent years rapid advances have taken place in the study ofchromosome structure and its relation to genetic content and DNAcomposition. In part, the progress has come in the form of improvedmethods of gene mapping based on the availability of large quantities ofpure DNA and RNA fragments for probes produced by genetic engineeringtechniques, e.g., Kao, “Somatic Cell Genetics and Gene Mapping,”International Review of Cytology, Vol. 85, pgs. 109-146 (1983), andD'Eustachio et al., “Somatic Cell Genetics in Gene Families,” Science,Vol. 220, pgs. 9, 19-924 (1983). The probes for gene mapping compriselabeled fragments of single-stranded or double-stranded DNA or RNA whichare hybridized to complementary sites on chromosomal DNA. With suchprobes it has been crucially important to produce pure, or homogeneous,probes to minimize hybridizations at locations other than at the site ofinterest, Henderson, “Cytological Hybridization to MammalianChromosomes,” International Review of Cytology, Vol. 76, pgs. 1-46(1982).

[0012] The hybridization process involves unravelling, or melting, thedouble-stranded nucleic acids of the probe and target by heating, orother means (unless the probe and target are single-stranded nucleicacids). This step is sometimes referred to as denaturing the nucleicacid. When the mixture of probe and target nucleic acids cool, strandshaving complementary bases recombine, or anneal. When a probe annealswith a target nucleic acid, the probe's location on the target can bedetected by a label carried by the probe or by some intrinsiccharacteristics of the probe or probe-target duplex. When the targetnucleic acid remains in its natural biological setting, e.g., DNA inchromosomes, mRNA in cytoplasm, portions of chromosomes or cell nuclei(albeit fixed or altered by preparative techniques), the hybridizationprocess is referred to as in situ hybridization.

[0013] In situ hybridization probes were initially limited toidentifying the location of genes or other well defined nucleic acidsequences on chromosomes or in cells. Comparisons of the mapping ofsingle-copy probes to normal and abnormal chromosomes were used toexamine chromosomal abnormalities. Cannizzaro et al., Cytogenetics andCell Genetics, 39:173-178 (1985). Distribution of the multiple bindingsites of repetitive probes could also be determined.

[0014] Hybridization with probes which have one target site in a haploidgenome, single-copy or unique sequence probes, has been used to map thelocations of particular genes in the genome [Harper and Saunders,“Localization of the Human Insulin Gene to the Distal End of the ShortArm of Chromosome 11, ” Proc. Natl. Acad. Sci., Vol. 78, pgs. 4458-4460(1981); Kao et al., “Assignment of the Structural Gene Coding forAlbumin to Chromosome 4,” Human Genetics, Vol. 62, pgs. 337-341 (1982)];but such hybridizations are not reliable when the size of the targetsite is small. As the amount of target sequence for low complexitysingle-copy probes is small, only a portion of the potential targetsites in a population of cells form hybrids with the probe. Therefore,mapping the location of the specific binding site of the probe has beencomplicated by background signals produced by non-specific binding ofthe probe and also by noise in the detection system (for example,autoradiography or immunochemistry). The unreliability of signals forsuch prior art single-copy probes has required statistical analysis ofthe positions of apparent hybridization signals in multiple cells to mapthe specific binding site of the probe.

[0015] Wallace et al., in “The Use of Synthetic Oligonucleotides asHybridization Probes. II. Hybridization of Oligonucleotides of MixedSequence to Rabbit Beta-Globin DNA,” Nucleic Acids Research, Vol. 9,pgs. 879-894 (1981), disclose the construction of syntheticoligonucleotide probes having mixed base sequences for detecting asingle locus corresponding to a structural gene. The mixture of basesequences was determined by considering all possible nucleotidesequences which could code for a selected sequence of amino acids in theprotein to which the structural gene corresponded.

[0016] Olsen et al., in “Isolation of Unique Sequence Human XChromosomal Deoxyribonucleic Acid,” Biochemistry, Vol. 19, pgs.2419-2428 (1980), disclose a method for isolating labeled uniquesequence human X chromosomal DNA by successive hybridizations: first,total genomic human DNA against itself so that a unique sequence DNAfraction can be isolated; second, the isolated unique sequence human DNAfraction against mouse DNA so that homologous mouse/human sequences areremoved; and finally, the unique sequence human DNA not homologous tomouse against the total genomic DNA of a human/mouse hybrid whose onlyhuman chromosome is chromosome X, so that a fraction of unique sequenceX chromosomal DNA is isolated. Individual clones are then isolated fromthis fraction and are candidates for human X chromosome specific DNAsequences.

[0017] Manuelidis et al., in “Chromosomal and Nuclear Distribution ofthe Hind III 1.9-KB Human DNA Repeat Segment,” Chromosoma, Vol .91, pp.28-38 (1984), disclose the construction of a single kind of DNA probefor detecting multiple loci on chromosomes corresponding to the locationof members of a family of repeated DNA sequences. Such probes are hereintermed repetitive probes.

[0018] Different repetitive sequences may have different distributionson chromosomes. They may be spread over all chromosomes as in the justcited reference, or they may be concentrated in compact regions of thegenome, such as, on the centromeres of the chromosomes, or they may haveother distributions. In some cases, such a repetitive sequence ispredominantly located on a single chromosome, and therefore is achromosome-specific repetitive sequence. [Willard et al., “Isolation andCharacterization of a Major Tandem Repeat Family from the Human XChromosome,” Nucleic Acids Research, Vol. 11, pgs. 2017-2033 (1983).]

[0019] A probe for repetitive sequences shared by all chromosomes can beused to discriminate between chromosomes of different species if thesequence is specific to one of the species. Total genomic DNA from onespecies which is rich in such repetitive sequences can be used in thismanner. [Pinkel et al. (III), PNAS USA, 83:2934 (1986); Manuelidis, Hum.Genet., 71:288 (1985) and Durnam et al., Somatic Cell Molec. Genet.,11:571 (1985.]

[0020] Recently, there has been an increased availability of probes forrepeated sequences (repetitive probes) that hybridize intensely andspecifically to selected chromosomes. [Trask et al., Hum. Genet., 78:251(1988) and references cited therein.] Such probes are now available forover half of the human chromosomes. In general, they bind to repeatedsequences on compact regions of the target chromosome near thecentromere. However, one probe has been reported that hybridizes tohuman chromosome 1p36, and there are several probes that hybridize tohuman chromosome Yq.

[0021] Hybridization with such probes permits rapid identification ofchromosomes in metaphase spreads, determination of the number of copiesof selected chromosomes in interphase nuclei [Pinkel et al. (I), PNASUSA, 83:2934 (1986); Pinkel et al. (II), Cold Spring Harbor Symp. Quant.Biol. 51:151 (1986) and Cremer et al., Hum. Genet, 74:346 (1986)] anddetermination of the relative positions of chromosomes in interphasenuclei [Trask et al., supra; Pinkel et al. (I), supra; Pinkel et al.(II), supra; Manuelidis, PNAS USA, 81:3123 (1984); Rappold et al., Hum.Genet., 67:317 (1984); Schardin et al., Hum. Genet., 71:282 (1985); andManuelidis, Hum. Genet., 71:288 (1985)].

[0022] However, many applications are still limited by the lack ofappropriate probes. For example, until the methods described herein wereinvented, probes with sufficient specificity for prenatal diagnosis werenot available for chromosome 13 or 21. Further, repetitive probes arenot very useful for detection of structural aberrations since theprobability is low that the aberrations will involve the region to whichthe probe hybridizes.

[0023] This invention overcomes the prior art limitations on the use ofprobes and dramatically enhances the application of in situhybridization for cytogenetic analysis. As indicated above, prior artprobes have not been useful for in-depth cytogenetic analysis. Lowcomplexity single-copy probes do not at this stage of hybridizationtechnology generate reliable signals. Although repetitive probes doprovide reliable signals, such signals cannot be tailored for differentapplications because of the fixed distribution of repetitive sequencesin a genome. The probes of this invention combine the hybridizationreliability of repetitive probes with the flexibility of being able totailor the binding pattern of the probe to any desired application.

[0024] The enhanced capabilities of the probes of this invention comefrom their increased complexity. Increasing the complexity of a probeincreases the probability, and therefore the intensity, of hybridizationto the target region, but also increases the probability of non-specifichybridizations resulting in background signals. However, within theconcept of this invention, it was considered that such backgroundsignals would be distributed approximately randomly over the genome.Therefore, the net result is that the target region could be visualizedwith increased contrast against such background signals.

[0025] Exemplified herein are probes in an approximate complexity rangeof from about 50,000 bases (50 kb) to hundreds of millions of bases.Such representative probes are for compact loci and whole humanchromosomes. Prior to this invention, probes employed for in situhybridization techniques had complexities below 40 kb, and moretypically on the order of a few kb.

[0026] Staining chromosomal material with the probes of this inventionis significantly different from the chemical staining of the prior art.The specificity of the probe produced staining of this invention arisesfrom an entirely new source—the nucleic acid sequences in a genome.Thus, staining patterns of this invention can be designed to highlightfundamental genetic information important to particular applications.

[0027] The procedures of this invention to construct probes of anydesired specificity provide significant advances in a broad spectrum ofcytogenetic studies. The analysis can be carried out on metaphasechromosomes and interphase nuclei. The techniques of this invention canbe especially advantageous for applications where high-quality bandingby conventional methods is difficult or suspected of yielding biasedinformation, e.g., in tumor cytogenetics. Reagents targeted to sites oflesions known to be diagnostically or prognostically important, such astumor type-specific translocations and deletions, among other tumorspecific genetic arrangements, permit rapid recognition of suchabnormalities. Where speed of analysis is the predominant concern, e.g.,detection of low-frequency chromosomal aberrations induced by toxicenvironmental agents, the compositions of this invention permit adramatic increase in detection efficiency in comparison to previoustechniques based on conventional chromosome banding.

[0028] Further, prenatal screening for disease-linked chromosomeaberrations (e.g., trisomy 21) is enhanced by the rapid detection ofsuch aberrations by the methods and compositions of this invention.Interphase aneuploidy analysis according to this invention isparticularly significant for prenatal diagnosis in that it yields morerapid results than are available by cell culture methods. Further, fetalcells separated from maternal blood, which cannot be cultured by routineprocedures and therefore cannot be analysed by conventional karyotypingtechniques, can be examined by the methods and compositions of thisinvention. In addition, the intensity, contrast and color combinationsof the staining patterns, coupled with the ability to tailor thepatterns for particular applications, enhance the opportunities forautomated cytogenetic analysis, for example, by flow cytometry orcomputerized microscopy and image analysis.

[0029] This application specifically claims chromosome specific reagentsfor the detection of genetic rearrangements and methods of using suchreagents to detect such rearrangements. Representative geneticrearrangements so detected are those that produce a fusiongene—BCR-ABL—that is diagnostic for chronic myelogenous leukemia (CML).

[0030] Chronic myelogenous leukemia (CML) is a neoplastic proliferationof bone marrow cells genetically characterized by the fusion of the BCRand ABL genes on chromosomes 9 and 22. That fusion usually involves areciprocal translocation t(9;22)(q34;q11), which produces thecytogenetically distinctive Philadelphia chromosome (Ph¹). However, morecomplex rearrangements may cause BCR-ABL fusion. At the molecular level,fusion can be detected by Southern analysis or by in vitro amplificationof the mRNA from the fusion gene using the polymerase chain reaction(PCR). Those techniques are sensitive but cannot be applied to singlecells.

[0031] Clearly, a sensitive method for detecting chromosomalabnormalities and, more specifically, genetic rearrangements, such as,for example, the tumor specific arrangements associated with CML, wouldbe a highly useful tool for genetic screening. This invention providessuch tools.

[0032] The following references are indicated in the ensuing text bynumbers as indicated:

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[0059] 27. D. Pinkel et al., Proc Natl Acad Sci USA 83,2934 (1986).

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[0061] 29. B. Trask and J. Hamlin, Genes and Development, 3:1913 (1989).

[0062] 30. J. B. Lawrence, C. A. Villnave and R. H. Singer, Cell 42,51(1988).

[0063] 31. G. D. Johnson and J. G. Nogueria J. Immunol. Methods 43, 349(1981).

[0064] 32. Hegewisch-Becker et al., J. Cell. Biochem. (Suppl.) 13E, 289(1989).

[0065] 33. Kohler et al., “Expression of BCR-ABL from TranscriptsFollowing Bone Marrow Transplant for Philadelphia Chromosome PositiveLeukemias”, (manuscript submitted).

[0066] 34. Heisterkamp et al., Nature, 315:758 (1985).

[0067] 35. Heisterkamp et al, J. Molec. Appl. Genet., 2:57 (1983).

[0068] Fusion of the proto-oncogene c-ABL from the long arm ofchromosome 9 with the BCR gene of chromosome 22 is a consistent findingin CML (1-3). That genetic change leads to formation of a BCR-ABLtranscript that is translated to form a 210kd protein present invirtually all cases of CML (4-6). In 90% of the cases, the fusion generesults from a reciprocal translocation involving chromosomes 9 and 22producing a cytogenetically distinct small acrocentric chromosome calledthe Philadelphia (Ph¹) chromosome (7-12), FIG. 8. However, standardcytogenetics does not have the resolution to distinguish closely spacedbreakpoints, such as those characteristic of CML and acute lymphocyticleukemia (ALL), and misses fusions produced by more complexrearrangements. Mapping and cloning of the breakpoint regions in bothgenes has lead to molecular techniques capable of demonstrating BCR-ABLfusion in CML cases where the Ph¹ chromosome could not be detectedcytogenetically (13-16). Southern analysis for BCR rearrangements hasbecome the standard for diagnosis of CML. More recently, fusion has beendetected by in vitro amplification of a cDNA transcript copied from CMLmRNA using reverse transcriptase (17-23). That technique permitsdetection of BCR-ABL transcript from CML cells present at lowfrequencies. Both of those techniques utilize nucleic acid obtained fromcell populations so that correlation between genotype and phenotype forindividual cells is not possible.

[0069] Described herein are chromosome-specific reagents and methods todetect genetic rearrangements, such as those exemplified herein for theBCR-ABL fusion, that supply information unavailable by existingtechniques.

SUMMARY OF THE INVENTION

[0070] This invention concerns methods of staining chromosomal materialbased upon nucleic acid sequence that employ one or more nucleic acidprobes. Said methods produce staining patterns that can be tailored forspecific cytogenetic analyses. It is further an object of this inventionto produce nucleic acid probes that are useful for cytogenetic analysis,that stain chromosomal material with reliable signals. Such probes areappropriate for in situ hybridization. Preferred nucleic acid probes forcertain applications of this invention are those of sufficientcomplexity to stain reliably each of two or more target sites.

[0071] The invention provides methods and compositions for stainingchromosomal material. The probe compositions of this invention at thecurrent state of hybridization techniques are typically of highcomplexity, usually greater than about 50 kb of complexity, thecomplexity depending upon the application for which the probe isdesigned. In particular, chromosome specific staining reagents areprovided which comprise heterogeneous mixtures of nucleic acidfragments, each fragment having a substantial fraction of its sequencessubstantially complementary to a portion of the nucleic acid for whichspecific staining is desired—the target nucleic acid, preferably thetarget chromosomal material. In general, the nucleic acid fragments arelabeled by means as exemplified herein and indicated infra. However, thenucleic add fragments need not be directly labeled in order for thebinding of probe fragments to the target to be detected; for example,such nucleic acid binding can be detected by anti-RNA/DNA duplexantibodies and antibodies to thymidine dimers. The nucleic acidfragments of the heterogenous mixtures include double-stranded orsingle-stranded RNA or DNA.

[0072] This invention concerns chromosome specific reagents and methodsof staining targeted chromosomal material that is in the vicinity of asuspected genetic earrangement. Such genetic rearrangement include butare not limited to translocations, inversions, insertions,amplifications and deletions. When such a genetic rearrangement isassociated with a disease, such chromosome specific reagents arereferred to as disease specific reagents or probes. When such a geneticrearrangement is associated with cancer, such reagents are referred toas tumor specific reagents or probes.

[0073] This invention provides for nucleic acid probes that reliablystain targeted chromosomal materials in the vicinity of one or moresuspected genetic rearrangements. Such nucleic acid probes useful forthe detection of genetic rearrangements are typically of highcomplexity. Such nucleic acid probes preferably comprise nucleic acidsequences that are substantially homologous to nucleic acid sequences inchromosomal regions that flank and/or extend partially or fully acrossbreakpoints associated with genetic rearrangements.

[0074] This invention further provides for methods and reagents todistinguish between cytogenetically similar but genetically differentchromosomal rearrangements.

[0075] Specifically herein exemplified are chromosome specific regentsand methods to detect genetic rearrangements, e.g., translocations,amplifications and insertions, that produce the BCR-ABL fusion which isdiagnostic for chronic myelogenous leukemia (CML). Such chromosomespecific reagents for the diagnosis of CML contain nucleic acidsequences which are substantially homologous to chromosomal sequences inthe vicinity of the translocation breakpoint regions of chromosomalregions 9q34 and 22q11 associated with CML.

[0076] Those reagents produce a staining pattern which is distinctivelyaltered when the BCR-ABL fusion characteristic of CML occurs. FIG. 11graphically demonstrates a variety of staining patterns which, alongwith other potential staining patterns, are altered in the presence of agenetic rearrangement, such as, the BCR-ABL fusion.

[0077] The presence of a genetic rearrangement can be determined byapplying the reagents of this invention according to methods hereindescribed and observing the proximity of and/or other characteristics ofthe signals of the staining patterns produced.

[0078] Preferably, the chromosome specific reagents used to detect CMLof this invention comprise nucleic acid sequences having a complexity offrom about 50 kilobases (kb) to about 1 megabase (Mb), more preferablyfrom about 50 kb to about 750 kb, and still more preferably from about200 kb to about 400 kb.

[0079] This invention further provides for methods of distinguishingbetween suspected genetic rearrangements that occur in relatively doseproximity in a genome wherein the chromosome specific reagents comprisenucleic add sequences substantially homologous to nucleic acid sequencesin the vicinity of said suspected genetic rearrangements. An example ofsuch a differentiation between two potential genetic rearrangements isthe differential diagnosis of CML from acute lymphocytic leukemeia(ALL).

[0080] This invention still further provides methods and reagents forproducing staining patterns in a patient who is afflicted with a diseaseassociated genetic rearrangement, such as those associated with theBCR-ABL fusion in CML, wherein said staining patterns are predictiveand/or indicative of the response of a patient to various therapeuticregimens, such as chemotherapy, radiation, surgery, and transplantation,such as bone marrow transplantation. Such staining patterns can beuseful in monitoring the status of such a patient, preferably on a cellby cell basis, and can be predictive of a disease recurrence for apatient that is in remission. Computer assisted microscopic analysis canassist in the interpretation of staining patterns of this invention, andthe invention provides for methods wherein computer assisted microscopicanalysis is used in testing patient cells on a call by cell basis, fore.g., to search for residual disease in a patient.

[0081] Still further, this invention provides for methods and reagentsto determine the molecular basis of genetic disease, and to detectspecific genetically based diseases.

[0082] Still further, this invention provides for methods and reagentsfor detecting contiguous gene syndromes comprising the in situhybridization of nucleic acid probes which comprise sequences which aresubstantially homologous to nucleic add sequences characteristic of oneor more components of a contiguous gene syndrome. Representative of sucha contiguous gene syndrome is Down syndrome.

[0083] Also provided are methods of simultaneously detecting geneticrearrangements of multiple loci in a genome comprising in situhybridization of high complexity nucleic acid probes comprising nucleicacid sequences that are substantially homologous to nucleic acidsequences in multiple loci in a genome.

[0084] Still further provided are methods of searching for geneticrearrangements in a genome. For example, conventional banding analysismay indicate an abnormality in a chromosomal region of a genome underexamination. Methods of this invention may include the application ofnucleic acid probes, produced from the vicinity of that chromosomalregion of a normal genome, by in situ hybridization to cells containingthe abnormality to detail the exact location and kind of geneticrearrangement of said abnormality by observation of the stainingpatterns so produced.

[0085] The invention still further provides for high complexity nucleicacid probes which have been optimized for rapid, efficient and automateddetection of genetic rearrangements.

[0086] One way to produce a probe of high complexity is to pool severalor many clones, for example, phage, plasmid, cosmid, and/or YAC clones,among others, wherein each clone contains an insert that is capable ofhybridizing to some part of the target in a genome. Another way toproduce such a probe is to use the polymerase chain reaction (PCR).

[0087] Heterogeneous in reference to the mixture of labeled nucleic acidfragments means that the staining reagents comprise many copies each offragments having different sequences and/or sizes (e.g., from thedifferent DNA clones pooled to make the probe). In preparation for use,these fragments may be cut, randomly or specifically, to adjust the sizedistribution of the pieces of nucleic add participating in thehybridization reaction.

[0088] As discussed more fully below, preferably the heterogeneous probemixtures are substantially free from nucleic acid sequences withhybridization capacity to non-target nucleic acid. Most of suchsequences bind to repetitive sequences which are shared by the targetand non-target nucleic acids, that is, shared repetitive sequences.

[0089] Methods to remove undesirable nucleic acid sequences and/or todisable the hybridization capacity of such sequences are discussed morefully below. [See Section II]. Such methods include but are not limitedto the selective removal or screening of shared repetitive sequencesfrom the probe; careful selection of nucleic acid sequences forinclusion in the probe; blocking shared repetitive sequences by theaddition of unlabeled genomic DNA, or, more carefully selecting nucleicacid sequences for inclusion in the blocking mixture; incubating theprobe mixture for sufficient time for reassociation of high copyrepetitive sequences, or the like.

[0090] Preferably, the staining reagents of the invention are applied tointerphase or metaphase chromosomal DNA by in situ hybridization, andthe chromosomes are identified or classified, i.e., karyotyped, bydetecting the presence of the label, such as biotin or ³H, on thenucleic acid fragments comprising the staining reagent.

[0091] The invention includes chromosome staining reagents for the totalgenomic complement of chromosomes, staining reagents specific to singlechromosomes, staining reagents specific to subsets of chromosomes, andstaining reagents specific to subregions within single or multiplechromosomes. The term “chromosome-specific,” is understood to encompassall of these embodiments of the staining reagents of the invention. Theterm is also understood to encompass staining reagents made from anddirected against both normal and abnormal chromosome types.

[0092] A preferred method of making the chromosome-specific stainingreagents of the invention includes: 1) isolating chromosomal DNA from aparticular chromosome type or target region or regions in the genome, 2)amplifying the isolated DNA to form a heterogeneous mixture of nucleicacid fragments, 3) disabling the hybridization capacity of or removingshared repeated sequences in the nucleic acid fragments, and 4) labelingthe nucleic acid fragments to form a heterogeneous mixture of labelednucleic acid fragments. As described more fully below, the ordering ofthe steps for particular embodiments varies according to the particularmeans adopted for carrying out the steps.

[0093] The present invention addresses problems associated withkaryotyping chromosomes, especially for diagnostic and dosimetricapplications. In particular, the invention overcomes problems whicharise because of the lack of stains that are sufficientlychromosome-specific by providing reagents comprising heterogeneousmixtures of nucleic acid fragments that can be hybridized to the targetDNA and/or RNA, e.g., the target chromosomes, target subsets ofchromosomes, or target regions of specific chromosomes. The stainingtechnique of the invention opens up the possibility of rapid and highlysensitive detection of chromosomal abnormalities, particularly geneticrearrangements, in both metaphase and interphase cells using standardclinical and laboratory equipment and improved analysis using automatedtechniques. It has direct application in genetic screening, cancerdiagnosis, and biological dosimetry.

[0094] This invention further specifically provides for methods andnucleic acid probes for staining fetal chromosomal material, whethercondensed, as in metaphase, or dispersed as in interphase. Stillfurther, the invention provides for a non-embryo-invasive method ofkaryotyping the chromosomal material of fetal cells, wherein the fetalcells have been separated from maternal blood. Such fetal cells arepreferably leukocytes and/or cytotrophoblasts. Exemplary nucleic acidprobes are high complexity probes chromosome-specific for chromosometypes 13,18 and/or 21. Representative probes comprisechromosome-specific Bluescribe plasmid libraries from which a sufficientnumber of shared repetitive sequences have been removed or thehybridization capacity thereof has been disabled prior to and/or duringhybridization with the target fetal chromosomes.

[0095] This invention still further provides for test kits comprisingappropriate nucleic acid probes for use in tumor cytogenetics, in thedetection of disease related loci, in the analysis of structuralabnormalities, for example translocations, among other geneticrearrangements, and for biological dosimetry.

[0096] This invention further provides for prenatal screening kitscomprising appropriate nucleic acid probes of this invention. Thisinvention also provides for test kits comprising high complexity probesfor the detection of genetic rearrangements, and specifically for thoseproducing the BCR-ABL fusion characteristic of CML.

[0097] The methods and compositions of this invention permit staining ofchromosomal material with patterns appropriate for a desiredapplication. The pattern may extend over some regions of one or morechromosomes, or over some or all the chromosomes of a genome and maycomprise multiple distinguishable sections, distinguishable, forexample, by multiple colors. Alternatively, the pattern may be focusedon a particular portion or portions of a genome, such as a portion orportions potentially containing a deletion or breakpoint that isdiagnostically or prognostically important for one or more tumors, or onthose portions of chromosomes having significance for prenataldiagnosis.

[0098] The staining patterns may be adjusted for the analysis methodemployed, for example, either a human observer or automated equipment,such as, flow cytometers or computer assisted microscopy. The patternsmay be chosen to be appropriate for analysis of condensed chromosomes ordispersed chromosomal material.

[0099] The invention further provides for automated means of detectingand analyzing chromosomal abnormalities, particularly geneticrearrangements, as indicated by the staining patterns produced accordingto this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0100]FIGS. 1A, B and C and FIGS. 2A and 2B illustrate the hybridizationof a chromosome-specific 21 library to human metaphase spread whereinthe inserts were cloned in Lambda phage Charon 21A. The hybridizationcapacity of the high copy repetitive sequences in the library wasreduced by the addition of unlabeled genomic DNA to the hybridizationmixture. The probe was labeled with biotin, which was detected withgreen FITC-avidin (fluorescein isothiocyanate avidin). All of the DNA inthe chromosomes was stained with the blue fluorescent dye DAPI(4,6-diamidino-2-phenylindole).

[0101]FIG. 1A is a binary image of the DAPI stain in the human metaphasespread obtained by using a TV camera attached to a fluorescencemicroscope. Filters appropriate for DAPI visualization were used.Computer processing of the image shows all portions above a chosenthreshold intensity as white, and the rest as black.

[0102]FIG. 1B is a binary image of the FITC staining of the same humanmetaphase spread as in FIG. 1A. The image was processed as in FIG. 1Abut the filter was changed in the microscope such that the FITC attachedto the probe is visible rather than the DAPI.

[0103]FIG. 1C is a binary image of the chromosome 21s alone,nonspecifically stained objects (which are smaller) having been removedby standard image processing techniques on the binary image of FIG. 1B.

[0104]FIG. 2A is a color photograph of the DAPI stain in a humanmetaphase spread which was prepared and hybridized contemporaneouslywith the spread shown in the computer generated binary images of FIGS.1A, B and C.

[0105]FIG. 2B is a color photograph of the fluorescein attached to theDNA probe in the same human metaphase spread as shown in FIG. 2A. It wasobtained by changing the filters in the fluorescence microscope toexcite fluorescein rather than DAPI. The photograph is comparable to thebinary image of FIG. 1B.

[0106]FIG. 3 is a photograph of a human metaphase spread prepared andhybridized contemporaneously with the spreads shown in FIGS. 1A, B and Cand 2A and B. The procedures used were the same except that PI(propidium iodide) instead of DAPI, was used to stain all thechromosomes. Both PI and fluorescein stains can be viewed with the samemicroscope filters. Color film was used such that the propidium iodidecounterstain appears red and the fluorescein of the probe appears yellowon the color film.

[0107]FIG. 4A shows the hybridization of the chromosome 4-specificlibrary in Bluescribe plasmids (the library pBS4) to a human metaphasespread wherein no unlabeled human genomic DNA was used, and wherein thehybridization mixture was applied immediately after denaturation. Bothcopies of chromosome 4 are seen as slightly brighter than the otherchromosomes. The small arrows indicate regions that are unstained withthe probe. As in FIG. 3 and as in the rest of the Figures below, PI isthe counterstain and fluorescein is used to label the probe.

[0108]FIG. 4B shows the hybridization of pBS-4 to a human metaphasespread wherein unlabeled human genomic DNA was used during thehybridization (Q=2 of genomic DNA; the meaning of Q is explained infra).Quantitative image analysis shows that the intensity per unit length ofthe chomosome 4s is about 20× that of the other chromosomes. Thechromosome 4s are yellow; the other chromosomes are red due to thepropidium iodide counterstain. Two layers of avidin-fluoresceinisothiocyanate have been used to make the target chromosomessufficiently bright to be measured accurately. However, the number 4chromosomes can be recognized easily after a single layer is applied.

[0109]FIG. 4C shows the same spread as in FIG. 4B but through a filterthat passes only the fluorescein isothiocyanate fluorescence.

[0110]FIG. 4D shows the detection of a radiation-induced translocation(arrows) involving chromosome 4s in a human metaphase spread whereinpBS-4 specific libraries are used. The contrast ratio is about 5×.

[0111]FIG. 4E shows that normal and two derivative chromosomes resultingfrom a translocation between chromosome 4 and 11 (in cell line RS4;11)can be detected by the compositions and methods of this invention ininterphase nuclei. They appear as three distinct domains.

[0112]FIG. 4F shows the hybridization of the chromosome 21-specificlibrary in Bluescribe plasmids (the library pBS-21) to a metaphasespread of a trisomy 21 cell line. A small amount of hybridization isvisible near the centromeres of the other acrocentric chromosomes.

[0113]FIG. 4G shows the same hybridization as in FIG. 4F but withinterphase nuclei. Clearly shown are the three chromosome 21 domains.

[0114]FIG. 4H shows the hybridization with a pool of 120 single copyprobes from chromosome 4 to a human metaphase spread. The number 4chromosomes are indicated by arrows.

[0115]FIG. 5 shows the hybridization of a yeast artificial chromosome(YAC) clone containing a 580 kb insert of human DNA to a human metaphasespread. A yellow fluorescein band on each of the chromosome 12s (at12q21.1) is visible against the propidium iodide counterstain.

[0116]FIG. 6 shows the hybridization of DNA from a human/hamster hybridcell containing one copy of human chromosome 19 to a human metaphasespread. A little to the right of the photograph's center are the twochromosome 19s which are brighter than the other chromosomes in thespread.

[0117]FIG. 7 illustrates a representative method of using the polymerasechain reaction (PCR) to produce probes of this invention which arereduced in repetitive sequences.

[0118]FIG. 8 illustrates the locations of probes to the CML breakpointand corresponding pattern of staining in both normal and CML metaphaseand interphase nuclei.

[0119] The left side shows schematic representations of the BCR gene onchromosome 22, the ABL gene of chromosome 9, and the BCR-ABL fusion geneon the Philadelphia chromosome. Also shown are the locations of CMLbreakpoints and their relation to the probes (32). The right showshybridization patterns expected for the c-hu-ABL and PEM12 probes tonormal and CML metaphase spreads and interphase nuclei.

[0120]FIG. 9 shows fluorescence in-situ hybridization (FISH) inmetaphase spreads and interphase nuclei. Panels A and B show ABL and BCRhybridization to normal metaphase spreads. The ABL signal (A) islocalized to the telomeric portion of 9q and the BCR signal (B) islocalized near the centromere of 22q. Panel C shows that ABL staining islocalized to the telomeric region of Philadelphia chromosome in a caseof CML with 46XY, t (9:22) (q34;q11). Panel D shows that ABL staining isinterstitial on the derivative 22 chromosome arising from an insertionalevent in a case of CML with 46XY ins (22:9)(q11;q34). Panel Eillustrates that the K562 cell line presents multiple signals localizedto a region of the interphase nucleus. Identical staining pattern wasseen with BCR probe indicating BCR-ABL fusion gene amplification. PanelF presents a metaphase spread from the K562 cell line showing fusiongene amplification localized to a single chromosome.

[0121]FIG. 10 illustrates fluorescence in-situ hybridization in CMLinterphase nuclei with ABL (red) and BCR (green) probes visualizedsimultaneously through a double band pass filter. Cells from a CMLpatient show the red-green (yellow) signals resulting from thehybridization to the BCR-ABL fusion gene and single red and greenhybridization signals to the normal BCR and ABL genes on chromosomes 22and 9.

[0122]FIG. 11 illustrates some exemplary probe strategies for detectionof structural aberrations. The design of the binding pattern, colorsetc., of the probe can be optimized for detection of geneticabnormalities in metaphase and/or interphase cells. Different patternsmay have advantages for particular applications. The drawings in FIG. 11illustrate some of the patterns useful for detection of someabnormalities. The examples are representative and not meant to beexhaustive; different patterns can be combined to allow for thedetection of multiple abnormalities in the same cell.

[0123] In the drawings of FIG. 11, the metaphase chromosomes are shownwith probe bound to both chromatids. The interphase nuclei are picturedto be in a stage of the cell cycle prior to replication of the portionof the chromosome to which the probe binds; thus there is only onechromatid for each interphase chromosome. When the probe binding isrestricted to only a portion of a chromosome, the signal is indicated aseither a black or white circle. Such a representation is employed toindicate different colors or otherwise distinguishable characteristicsof the staining. Patterns containing more than two distinguishablecharacteristics (three colors, different ratios of colors etc.) permitmore complex staining patterns than those illustrated. Chromosomallocations of the breakpoints in the DNA are indicated with horizontallines next to the abnormal chromosomes.

[0124] a. Section a) represents the use of a probe which stains a wholechromosome. Such a probe can be used to detect a translocation thatoccurs anywhere along the chromosome. The color photograph of FIG. 12shows use of such a stain for chromosome 22 to detect a translocation,in this case that which occurs with CML. Such an approach to staining isnot very useful in interphase nuclei since the region of the nucleusthat is stained is relatively large; overlaps in the stained regions canmake interpretation difficult in many nuclei.

[0125] b. Section b) represents the reduction of the stained region ofthe chromosome shown in a) to that in the vicinity of a breakpoint,providing information focused on events in that region. The stainingpattern can be continuous or discontinuous across the breakpoint, justso that some binding is on both sides of the breakpoint. Such a stainingpattern requires only one “color”, but gives no information about whichother genomic region may be involved in the exchange.

[0126] c. Section c) represents the use of a probe which binds tosequences which come together as a result of the rearrangement andallows for the detection in metaphase and interphase cells. In this casethe different sequences are stained with different “colors”. Such astaining pattern is that used in the examples of Section VIII of thethis application.

[0127] d. Section d) represents an extension of c) by including stainingof both sides of both breakpoints involved in the rearrangement.Different “colors” are used as indicated. The additional informationsupplied by the more complex staining pattern may assist withinterpretation of the nuclei. It might also permit recognition of anapparent insertional event as discussed herein.

[0128] e. Section e) represents the detection of an inversion in onehomologue of a chromosome.

[0129] f. Section f) represents a staining pattern useful in thedetection of a deletion. A deletion could also be detected with a probethat stains only the deleted region; however, lack of probe binding maybe due to reasons other than deletion of the target sequence. Theflanking regions stained a different “color” serve as controls forhybridization.

[0130]FIG. 12 illustrates a staining pattern to detect a rearrangementby staining a whole chromosome, in this case a rearrangement ofchromosome 22 associated with CML. The metaphase spread of this figureis from a CML cell that has been stained with a probe which binds allalong chromosome 22. Probe-stained regions appear yellow. The rest ofthe DNA has been stained with the red-fluorescing chemical stainpropidium iodide. The entirely yellow chromosome is a normal copy ofchromosome 22. Just below said normal chromosome 22 is the Philadelphiachromosome, a small part yellow and part red chromosome. Below and tothe right of the Philadelphia chromosome is the abnormal chromosome 9(red) with the distal part of chromosome 22 (yellow) attached. Thephotograph of this figure illustrates the staining pattern representedin part a) of the previous figure.

DETAILED DESCRIPTION OF THE INVENTION

[0131] This invention concerns the use of nucleic acid probes to staintargeted chromosomal material in patterns which can extend along one ormore whole chromosomes, and/or along one or more regions on one or morechromosomes, including patterns which extend over an entire genome. Thestaining reagents of this invention facilitate the microscopic and/orflow cytometric identification of normal and aberrant chromosomes andprovide for the characterization of the genetic nature of particularabnormalities, such as, genetic rearrangements. The term“chromosome-specific” is herein defined to encompass the terms “targetspecific” and “region specific”, that is, when the staining compositionis directed to one chromosome, it is chromosome-specific, but it is alsochromosome-specific when it is directed, for example, to multipleregions on multiple chromosomes, or to a region of only one chromosome,or to regions across the entire genome. The term chromosome-specificoriginated from the use of recombinant DNA libraries made by cloning DNAfrom a single normal chromosome type as the source material for theinitial probes of this invention. Libraries made from DNA from regionsof one or more chromosomes are sources of DNA for probes for that regionor those regions of the genome. The probes produced from such sourcematerial are region-specific probes but are also encompassed within thebroader phrase “chromosome-specific” probes. The term “target specific”is interchangeably used herein with the term “chromosome-specific”.

[0132] The word “specific” as commonly used in the art has two somewhatdifferent meanings. The practice is followed herein. “Specific” mayrefer to the origin of a nucleic acid sequence or to the pattern withwhich it will hybridize to a genome as part of a staining reagent. Forexample, isolation and cloning of DNA from a specified chromosomeresults in a “chromosome-specific library”. [Eg., Van Dilla et al.,“Human Chromosome-Specific DNA Libraries: Construction andAvailability,” Biotechnology, 4:537 (1986).] However, such a librarycontains sequences that are shared with other chromosomes. Such sharedsequences are not chromosome-specific to the chromosome from which theywere derived in their hybridization properties since they will bind tomore than the chromosome of origin. A sequence is “chromosome-specific”if it binds only to the desired portion of a genome. Such sequencesinclude single-copy sequences contained in the target or repetitivesequences, in which the copies are contained predominantly in thetarget.

[0133] “Chromosome-specific” in modifying “staining reagent” refers tothe overall hybridization pattern of the nucleic acid sequences thatcomprise the reagent. A staining reagent is chromosome-specific ifuseful contrast between the target and non-target chromosomal materialis achieved (that is, that the target can be adequately visualized).

[0134] A probe is herein defined to be a collection of nucleic acidfragments whose hybridization to the target can be detected. The probeis labeled as described below so that its binding to the target can bevisualized. The probe is produced from some source of nucleic addsequences, for example, a collection of clones or a collection ofpolymerase chain reaction (PCR) products. The source nucleic acid maythen be processed in some way, for example, by removal of repetitivesequences or blocking them with unlabeled nucleic acid withcomplementary sequence, so that hybridization with the resulting probeproduces staining of sufficient contrast on the target. Thus, the wordprobe may be used herein to refer not only to the detectable nucleicacid, but also to the detectable nucleic add in the form in which it isapplied to the target, for example, with the blocking nucleic acid, etc.The blocking nucleic acid may also be mentioned separately. What “probe”refers to specifically should be clear from the context in which theword is used.

[0135] When two or more nucleic add probes of this invention are mixedtogether, they produce a new probe which when hybridized to a targetaccording to the methods of this invention, produces a staining patternthat is a combination of the staining patterns individually produced bythe component probes thereof. Thus, the terms “probe” and “probes” (thatis, the singular and plural forms) can be used interchangeably withinthe context of a staining pattern produced. For example, if one probe ofthis invention produces a dot on chromosome 9, and another probeproduces a band on chromosome 11, together the two probes form a probewhich produces a dot/band staining pattern.

[0136] The term “labeled” is herein used to indicate that there is somemethod to visualize the bound probe, whether or not the probe directlycarries some modified constituent. Section III infra describes variousmeans of directly labeling the probe and other labeling means by whichthe bound probe can be detected.

[0137] The terms “staining” or “painting” are herein defined to meanhybridizing a probe of this invention to a genome or segment thereof,such that the probe reliably binds to the targeted chromosomal materialtherein and the bound probe is capable of being visualized. The terms“staining” or “painting” are used interchangeably. The patternsresulting from “staining” or “painting” are useful for cytogeneticanalysis, more particularly, molecular cytogenetic analysis. Thestaining patterns facilitate the microscopic and/or flow cytometricidentification of normal and abnormal chromosomes and thecharacterization of the genetic nature of particular abnormalities.Section III infra describes methods of rendering the probe visible.Since multiple compatible methods of probe visualization are available,the binding patterns of different components of the probe can bedistinguished-for example, by color. Thus, this invention is capable ofproducing any desired staining pattern on the chromosomes visualizedwith one or more colors (a multi-color staining pattern) and/or otherindicator methods. The term “staining” as defined herein does notinclude the concept of staining chromosomes with chemicals as inconventional karotyping methods although such conventional stains may beused in conjunction with the probes of this invention to allowvisualization of those parts of the genome where the probe does notbind. The use of DAPI and propidium iodide for such a purpose isillustrated in the figures.

[0138] The phrase “high complexity” is defined herein to mean that theprobe, thereby modified contains on the order of 50,000 (50 kb) orgreater, up to many millions or several billions, of bases of nucleicacid sequences which are not repeated in the probe. For example,representative high complexity nucleic acid probes of this invention canhave a complexity greater than 50 kb, greater than 100,000 bases (100kb), greater than 200,000 (200 kb), greater than 500,000 bases (500 kb),greater than one million bases (1 Mb), greater than 2 Mb, greater than10 Mb, greater than 100 Mb, greater than 500 Mb, greater than 1 billionbases and still further greater than several billion bases.

[0139] The term “complexity” is defined herein according to the standardfor nucleic acid complexity as established by Britten et al., Methods ofEnzymol., 29:363 (1974). See also Cantor and Schimmel, BiophysicalChemistry: Part III: The Behavior of Biological Macromolecules, at1228-1230 (Freeman and Co. 1980) for further explanation andexemplification of nucleic acid complexity.

[0140] The complexity preferred for a probe composition of thisinvention is dependent upon the application for which it is designed. Ingeneral, the larger the target area, the more complex is the probe. Itis anticipated that the complexity of a probe needed to produce adesired pattern of landmarks on a chromosome will decrease ashybridization sensitivity increases, as progress is made inhybridization technology. As the sensitivity increases, the reliabilityof the signal from smaller target sites will increase. Therefore,whereas from about a 40 kb to about a 100 kb target sequence may bepresently necessary to provide a reliable, easily detectable signal,smaller target sequences should provide reliable signals in the future.Therefore, as hybridization sensitivity increases, a probe of a certaincomplexity, for example, 100 kb, should enable the user to detectconsiderably more loci in a genome than are presently reliably detected;thus, more information will be obtained with a probe of the samecomplexity. The term “complexity” therefore refers to the complexity ofthe total probe no matter how many visually distinct loci are to bedetected, that is, regardless of the distribution of the target sitesover the genome.

[0141] As indicated above, with current hybridization techniques it ispossible to obtain a reliable, easily detectable signal with a probe ofabout 40 kb to about 100 kb (eg. the probe insert capacity of one or afew cosmids) targeted to a compact point in the genome. Thus, forexample, a complexity in the range of approximately 100 kb now permitshybridization to both sides of a tumor-specific translocation. Theportion of the probe targeted to one side of the breakpoint can belabeled differently from that targeted to the other side of thebreakpoint so that the two sides can be differentiated with differentcolors, for example. Proportionately increasing the complexity of theprobe permits analysis of multiple compact regions of the genomesimultaneously. The conventional banding patterns produced by chemicalstains may be replaced according to this invention with a series ofprobe-based, color coded (for example), reference points along eachchromosome or significant regions thereof.

[0142] Uniform staining of an extended contiguous region of a genome,for example, a whole chromosome, requires a probe complexityproportional to but substantially less than, the complexity of thetarget region. The complexity required is only that necessary to providea reliable, substantially uniform signal on the target. Section V.B,infra, demonstrates that fluorescent staining of human chromosome 21,which contains about 50 megabases (Mb) of DNA, is sufficient with aprobe complexity of about 1 Mb. FIG. 4H illustrates hybridization ofabout 400 kb of probe to human chromosome 4, which contains about 200 Mbof DNA. In that case, gaps between the hybridization of individualelements of the probe are visible. FIGS. 4B and 4F demonstrate theresults achieved with probes made up of entire libraries for chromosomes4 and 21, respectively. The chromosomes are stained much more densely asshown in FIGS. 4B and 4F than with the lower complexity probe comprisingsingle-copy nucleic acid sequences used to produce the pattern of FIG.4H.

[0143] Increasing the complexity beyond the minimum required foradequate staining is not detrimental as long as the total nucleic acidconcentration in the probe remains below the point where hybridizationis impaired. The decrease in concentration of a portion of a sequence inthe probe is compensated for by the increase in the number of targetsites. In fact, when using double-stranded probes, it is preferred tomaintain a relatively low concentration of each portion of sequence toinhibit reassociation before said portion of sequence can find a bindingsite in the target.

[0144] The staining patterns of this invention comprise one or more“bands”. The term “band” is herein defined as a reference point in agenome which comprises a target nucleic acid sequence bound to a probecomponent, which duplex is detectable by some indicator means, and whichat its narrowest dimension provides for a reliable signal under theconditions and protocols of the hybridization and the instrumentation,among other variables, used. A band can extend from the narrow dimensionof a sequence providing a reliable signal to a whole chromosome tomultiple regions on a number of chromosomes.

[0145] The probe-produced bands of this invention are to bedistinguished from bands produced by chemical staining as indicatedabove in the Background. The probe-produced bands of this invention arebased upon nucleic acid sequence whereas the bands produced by chemicalstaining depend on natural characteristics of the chromosomes, but notthe actual nucleic add sequence. Further, the banding patterns producedby chemical staining are only interpretable in terms of metaphasechromosomes whereas the probe-produced bands of this invention areuseful both for metaphase and interphase chromosomes.

[0146] One method of forming the probes of the present invention is topool many different low complexity probes. Such a probe would thencomprise a “heterogeneous mixture” of individual cloned sequences. Thenumber of clones required depends on the extent of the target area andthe capacity of the cloning vector. If the target is made up of severaldiscrete, compact loci, that is, single spots at the limit ofmicroscopic resolution, then about 40 kb, more preferably 100 kb, foreach spot gives a reliable signal given current techniques. The portionof the probe for each spot may be made up from, for example, a singleinsert from a yeast artificial chromosome (YAC), from several cosmidseach containing 35-40 kb or probe sequence, or from about 25 plasmidseach with 4 kb of sequence.

[0147] Representative heterogeneous mixtures of clones exemplifiedherein include phage (FIGS. 1, 2 and 3), and plasmids (FIG. 4). Yeastartificial chromosomes (YACS) (FIG. 5), and a single human chromosome inan inter-species hybrid cell (FIG. 6) are examples of high complexityprobes for single loci and an entire chromosome that can be propagatedas a single clone.

[0148] A base sequence at any point in the genome can be classified aseither “single-copy” or “repetitive”. For practical purposes thesequence needs to be long enough so that a complementary probe sequencecan form a stable hybrid with the target sequence under thehybridization conditions being used. Such a length is typically in therange of several tens to hundreds of nucleotides.

[0149] A “single-copy sequence” is that wherein only one copy of thetarget nucleic acid sequence is present in the haploid genome.“Single-copy sequences” are also known in the art as “unique sequences”.A “repetitive sequence” is that wherein there are more than one copy ofthe same target nucleic acid sequence in the genome. Each copy of arepetitive sequence need not be identical to all the others. Theimportant feature is that the sequence be sufficiently similar to theother members of the family of repetitive sequences such that under thehybridization conditions being used, the same fragment of probe nucleicacid is capable of forming stable hybrids with each copy. A “sharedrepetitive sequence” is a sequence with some copies in the target regionof the genome, and some elsewhere.

[0150] When the adjectives “single-copy”, “repetitive”, “sharedrepetitive”, among other such modifiers, are used to describe sequencesin the probe, they refer to the type of sequence in the target to whichthe probe sequence will bind. Thus, “a repetitive probe” is one thatbinds to a repetitive sequence in the target; and “a single-copy probe”binds to a single-copy target sequence.

[0151] Repetitive sequences occur in multiple copies in the haploidgenome. The number of copies can range from two to hundreds ofthousands, wherein the Alu family of repetitive DNA are exemplary of thelatter numerous variety. The copies of a repeat may be clustered orinterspersed throughout the genome. Repeats may be clustered in one ormore locations in the genome, for example, repetitive sequencesoccurring near the centromeres of each chromosome, and variable numbertandem repeats (VNTRs) [Nakamura et al, Science, 235:1616 (1987)]; orthe repeats may be distributed over a single chromosome [for example,repeats found only on the X chromosome as described by Bardoni et al.,Cytogenet. Cell Genet., 46:575 (1987)]; or the repeats may bedistributed over all the chromosomes, for example, the Alu family ofrepetitive sequences.

[0152] Herein, the terms repetitive sequences, repeated sequences andrepeats are used interchangeably.

[0153] Shared repetitive sequences can be clustered or interspersed.Clustered repetitive sequences include tandem repeats which are so namedbecause they are contiguous on the DNA molecule which forms the backboneof a chromosome. Clustered repeats are associated with well-definedregions of one or more chromosomes, e.g., the centromeric region. If oneor more clustered repeats form a sizable fraction of a chromosome, andare shared with one or more non-target regions of the genome and areconsequently removed from the heterogeneous mixture of fragmentsemployed in the invention or the hybridization capacity thereof isdisabled, perfect uniformity of staining of the target region may not bepossible. That situation is comprehended by the use of the term“substantially uniform” in reference to the binding of the heterogeneousmixture of labeled nucleic acid fragments to the target.

[0154] Chromosome-specific staining of the current invention isaccomplished by using nucleic acid fragments that hybridize to sequencesspecific to the target. These sequences may be either single-copy orrepetitive, wherein the copies of the repeat occur predominantly in thetarget area. FIG. 4H and the results of the work detailed in section Vinfra indicate that probes can be made of single-copy sequences.However, in probes such as that of FIG. 4B, low-copy chromosome-specificrepeats [Nakamura et al., and Bardoni et al., supra] may contribute tothe hybridization as well.

[0155] If nucleic acid fragments complementary to non-target regions ofthe genome are included in the probe, for example, shared repetitivesequences or non-specific sequences, their hybridization capacity needsto be sufficiently disabled or their prevalence sufficiently reduced, sothat adequate staining contrast can be obtained. Section V and FIG. 4Hshow examples of hybridization with probes that contain pools of clonesin which each clone has been individually selected so that it hybridizesto single-copy sequences or very low copy repetitive sequences. Theremaining figures illustrate use of probes that contain fragments thatcould have hybridized to high-copy repetitive sequences, but which havehad the hybridization capacity of such sequences disabled.

[0156] The nucleic acid probes of this invention need not be absolutelyspecific for the targeted portion of the genome. They are intended toproduce “staining contrast”. “Contrast” is quantified by the ratio ofthe stain intensity of the target region of the genome to that of theother portions of the genome For example, a DNA library produced bycloning a particular chromosome, such as those listed in Table I, can beused as a probe capable of staining the entire chromosome. The librarycontains sequences found only on that chromosome, and sequences sharedwith other chromosomes. In a simplified (approximately true to life)model of the human genome, about half of the chromosomal DNA falls intoeach class. If hybridization with the whole library were capable ofsaturating all of the binding sites, the target chromosome would betwice as bright (contrast ratio of 2) as the others since it wouldcontain signal from the specific and shared sequences in the probe,whereas the other chromosome would only have signal from the sharedsequences. Thus, only a modest decrease in hybridization of the sharedsequences in the probe would substantially enhance the contrast.Contaminating sequences which only hybridize to non-targeted sequences,for example, impurities in a library, can be tolerated in the probe tothe extent that said sequences do not reduce the staining contrast belowuseful levels.

[0157] In reality all of the target sites may not be saturated duringthe hybridization, and many other mechanisms contribute to producingstaining contrast, but this model illustrates one general considerationin using probes targeted at a large portion of a genome.

[0158] The required contrast depends on the application for which theprobe is designed. When visualizing chromosomes and nuclei, etc.,microscopically, a contrast ratio of two or greater is often sufficientfor identifying whole chromosomes. In FIGS. 4D-F, the contrast ratio is3-5. The smaller the individual segments of the target region, thegreater the contrast needs to be to permit reliable recognition of thetarget relative to the fluctuations in staining of the non-targetedregions. When quantifying the amount of target region present in a cellnucleus by fluorescence intensity measurements using flow cytometry orquantitative microscopy, the required contrast ratio is on the order of1/T or greater on average for the genome, where T is the fraction of thegenome contained in the targeted region. When the contrast ratio isequal to 1/T, half of the total fluorescence intensity comes from thetarget region and half from the rest of the genome. For example, whenusing a high complexity probe for chromosome 1, which comprises about10% of the genome, the required contrast ratio is on the order of 10,that is, for the chromosome 1 fluorescence intensity to equal that ofthe rest of the genome.

[0159] Background staining by the probe, that is, to the non-targetregion of the genome, may not be uniform. FIG. 4F shows that achromosome 21 specific probe contains probe fragments that hybridizeweakly to compact regions near the centromeres of other acrocentrichuman chromosomes. This degree of non-specificity does not inhibit itsuse in the illustrated applications. For other applications, removal ofor further disabling the hybridization capacity of the probe fragmentsthat bind to these sequences may be necessary.

[0160] For other applications, repetitive sequences that bind tocentromeres, for example, alpha-satellite sequences, and/or telomerescan be part of the chromosome-specific staining reagents wherein thetarget includes some or all of the centromeres and/or telomeres in agenome along with perhaps other chromosomal regions. Exemplary of suchan application would be that wherein the staining reagent is designed todetect random structural aberrations caused by dastogenic agents thatresult in dicentric chromosomes and other structural abnormalities, suchas translocations. Addition of sequences which bind to all centromeresin a genome, for example to the probe used to create the stainingpattern of FIG. 4D, would allow more reliable distinguishing betweendicentrics and translocations.

[0161] Application of staining reagents of this invention to a genomeresults in a substantially uniform distribution of probe hybridized tothe targeted regions of a genome. The distribution of bound probe isdeemed “substantially uniform” if the targeted regions of the genome canbe visualized with useful contrast. For example, a target issubstantially uniformly stained in the case wherein it is a series ofvisually separated loci if most of the loci are visible in most of thecells.

[0162] “Substantial proportions” in reference to the base sequences ofnucleic acid fragments that are comple- mentary to chromosomal DNA meansthat the complementarity is extensive enough so that the fragments formstable hybrids with the chromosomal DNA under the hybridizationconditions used. In particular, the term comprehends the situation wherethe nucleic acid fragments of the heterogeneous mixture possess someregions of sequence that are not perfectly complementary to targetchromosomal material. The stringency can be adjusted to control theprecision of the complementarity required for hybridization.

[0163] The phrase “metaphase chromosomes” is herein defined to mean notonly chromosomes condensed in the metaphase stage of mitosis butincludes any condensed chromosomes, for example, those condensed bypremature chromosome condensation.

[0164] To disable the hybridization capacity of a nucleic acid sequenceis herein sometimes abbreviated as “disabling the nucleic acidsequence”.

[0165] The methods and reagents of this invention find a particularlyappropriate application in the field of diagnostic cytogenetics,particularly in the field of diagnostic interphase cytogenetics.Detecting genetic rearrangements that are associated with a disease,such as cancer, are a specific application of the chromosome specificreagents and staining methods of this invention.

[0166] Contiguous gene syndromes are an example of the geneticrearrangements that the probes and methods of this invention canidentify. Contiguous gene syndromes are characterized by the presence ofseveral closely spaced genes which are in multiple and/or reduced copynumber. Down syndrome is an example of a contiguous gene syndromewherein an extra copy of a chromosomal region containing several genesis present.

[0167] Particularly described herein is the application of chromosomespecific reagents and methods for detecting genetic rearrangements thatproduce the BCR-ABL fusion associated with CML. Such reagents areexemplary of disease specific, in this case tumor specific, probes whichcan be labeled, directly and/or indirectly, such that they arevisualizable when bound to the targeted chromosomal material, which inthe case of CML, is the vicinity of the translocation breakpoint regionsof chromosomal regions 9q34 and 22q11 known to be associated with CML.In the examples provided in Section VIII of this application, the probesare labeled such that a dual color fluorescence is produced in thestaining pattern of said probes upon in situ hybridization [fluorescentin situ hybridication (FISH)]; however, staining patterns can beproduced in many colors as well as other types of signals, and anyvisualization means to signal the probe bound to its target can be usedin the methods of this invention.

[0168] Section VIII herein describes representative methods and reagentsof this invention to detect genetic rearrangements. The examples ofSection VIII concern genetic rearrangements that produce the BCR-ABLfusion that is characteristic of CML. The approach in such examples isbased on FISH with probes from chromosomes 9 and 22 that flank the fusedBCR and ABL sequences in essentially all cases of CML (FIG. 8). Theprobes when hybridized to the chromosomal material of both normal andabnormal cells produce staining patterns that are different asillustrated in FIGS. 8-12. The staining patterns produced by suchexemplary probes are different in normal and abnormal cells; thestaining pattern present when the genetic rearrangement occurs isdistinctively altered from that of the staining pattern shown byhybridizing the probes to chromosomal material that does not contain thegenetic rearrangement. Further, staining patterns are distinctivelydifferent for one type of genetic rearrangement versus another. Forexample, the staining patterns produced upon hybridization of nucleicacid probes of this invention to chromosomal material containing agenetic rearrangement associated with ALL is distinctively differentfrom that produced upon hybridization of such probes to chromosomalmaterial containing the BCR-ABL fusion characteristic of CML. Thus, themethods and reagents of this invention provide for differentialdiagnosis of related diseases.

[0169] The examples of Section VIII provide for the diagnosis of CMLbased upon the proximity of the fluorescent signals in the stainingpatterns, and rely upon a 1 micron cutoff point for determination of thepresence of a fusion. The proximity distance of signals is only onecharacteristic, among many others, of signals that can be used to detectthe presence of a genetic rearrangement. Further, the proximity distanceis dependent on the particular cell preparation techniques employed andthe size of the nuclei therein, and for a particular cell preparation isrelative depending on the distance between signals in normal andabnormal cells.

[0170] The staining patterns exemplified in the examples of Section VIIIare representative of one type of probe strategy. Many other probestrategies can be employed. FIG. 11 illustrates some other exemplaryprobe strategies for detecting genetic rearrangements, the patterns ofwhich can be modified and optimized and otherwise varied to detectparticular genetic rearrangements.

[0171] Use of other disease specific reagents of this invention would beanalogous to the methods detailed in Section VIII for CML. For example,the diagnosis and study of acute lymphocytic leukemia (ALL) may beaccomplished by replacing the BCR probe (PEM12) of Section VIII with aprobe from the 5′ end of the BCR gene. ALL is of particular interestbecause the Ph′ chromosome is the most common cytogenetic abnormality inthat disease, and the presence of such a chromosome is indicative of avery aggressive neoplasm.

[0172] The methods and reagents herein exemplified, particularly inSection VIII, provide for the means to distinguish betweencytogenetically similar but genetically different diseases.“Cytogenetically” in that particular context refers to a similaritydetermined by conventional banding analysis. CML and ALL are in thatcontext cytogenetically similar in that conventional banding analysiscan not distinguish them because the breakpoints associated with eachare so dose together in the human genome.

[0173] Further, this invention provides methods and reagents that can beused in a cytogenetic research mode for the study of the molecular basesof genetic disease. For example, if an abnormality in a person'skaryotype is noted by conventional banding analysis, the probes andreagents of this invention can be used to detect any geneticrearrangements in the vicinity of said abnormality. The underlyingmolecular basis of the abnormality can be determined by the methods andreagents of this invention, and the resulting differences at the geneticlevel may be indicative of different treatment plans and prognosticallyimportant. The underlying genetic rearrangements may be found to beconsistently associated with a set of phenotypic characteristics in apopulation.

[0174] The following sections provide examples of making and using thestaining compositions of this invention and are for purposes ofillustration only and not meant to limit the invention in any way. Thefollowing abbreviations are used.

ABBREVIATIONS

[0175] BN—bicarbonate buffer with NP-40

[0176] DAPI—4,6-diamidino-2-phenylindole

[0177] DCS—as in fluorescein-avidin DCS (a commercially available cellsorter grade of fluorescein Avidin D)

[0178] AAF—N-acetoxy-N-2-acetyl-aminofluorene

[0179] EDTA—ethylenediaminetetraacetate

[0180] FACS—fluorescence-activated cell sorting

[0181] FITC—fluorescein isothiocyanate

[0182] IB—isolation buffer

[0183] NP-40—non-ionic detergent commercially available from Sigma asNonidet P-40 (St. Louis, Mo.)

[0184] PBS—phosphate-buffered saline

[0185] PI—propidium iodide

[0186] PMSF—phenylmethylsulfonyl fluoride

[0187] PN—mixture of 0.1 M NaH₂PO₄ and 0.1 M buffer Na₂HPO₄, pH8; 0.1%NP-40

[0188] PNM—Pn buffer plus 5% nonfat dry milk (centrifuged); buffer 0.02%Na azide

[0189] SDS—sodium dodecyl sulfate

[0190] SSC—0.15 M NaC1/0.015 M Na citrate, pH 7

[0191] VNTR—variable number tandem repeat

I. METHODS OF PREPARING CHROMOSOME-SPECIFIC STAINING REAGENTS I.A.Isolation of Chromosome-Specific DNA and Formation of DNA FragmentLibraries

[0192] The first step in a preferred method of making the compositionsof the invention is isolating chromosome-specific DNA (which termincludes target-specific and/or region-specific DNA, as indicated above,wherein specific refers to the origin of the DNA). This step includesfirst isolating a sufficient quantity of the particular chromosome typeor chromosomal subregion to which the staining composition is directed,then extracting the DNA from the isolated chromosome(s) or chromosomalsubregion(s). Here “sufficient quantity” means sufficient for carryingout subsequent steps of the method. Preferably, the extracted DNA isused to create a library of DNA inserts by cloning using standardgenetic engineering techniques.

[0193] Preferred cloning vectors include, but are not limited to, yeastartificial chromosomes (YACS), plasmids, bacteriophages and cosmids.Preferred plasmids are Bluescribe plasmids; preferred bacteriophages arelambda insertion vectors, more preferably Charon 4A, Charon 21A, Charon35, Charon 40 and GEM11; and preferred cosmids include Lawrist 4,Lawrist 5 and sCos1.

[0194] As indicated above, the DNA can be isolated from any source.

[0195] Chromosome-specific staining reagents can be made from both plantand animal DNA according to the methods of this invention. Importantsources of animal DNA are mammals, particularly primates or rodentswherein primate sources are more particularly human and monkey, androdent sources are more particularly rats or mice, and more particularlymice.

[0196] 1. Isolating DNA from an Entire Chromosome. A preferred means forisolating particular whole chromosomes (specific chromosome types) is bydirect flow sorting [fluorescence-activated cell sorting (FACS)] ofmetaphase chromosomes with or without the use of interspecific hybridcell systems. For some species, every chromosome can be isolated bycurrently available sorting techniques. Most, but not all, humanchromosomes are currently isolatable by flow sorting from human cells,Carrano et al., “Measurement and Purification of Human Chromosomes byFlow Cytometry and Sorting,” Proc. Natl. Acad. Sd., Vol. 76, pgs.1382-1384 (1979). Thus, for isolation of some human chromosomes, use ofthe human/rodent hybrid cell system may be necessary, see Kao, “SomaticCell Genetics and Gene Mapping,” International Review of Cytology., Vol.85, pgs. 109-146 (1983), for a review, and Gusella et al., “Isolationand Localization of DNA Segments from Specific Human Chromosomes,” Proc.Natl. Acad. Sci., Vol. 77, pgs. 2829-2833 (1980). Chromosome sorting canbe done by commercially available fluorescence-activated sortingmachines, e.g., Becton Dickinson FACS-II, Coulter Epics V sorter, orspecial purpose sorters optimized for chromosome sorting or likeinstrument.

[0197] DNA is extracted from the isolated chromosomes by standardtechniques, e.g., Marmur, “A Procedure for the Isolation ofDeoxyribonucleic Acid from Micro-Organisms,” J. Mol. Biol., Vol. 3, pgs.208-218 (1961); or Maniatis et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory, 1982) pgs. 280-281. Thesereferences are incorporated by reference for their descriptions of DNAisolation techniques.

[0198] Generation of insert libraries from the isolatedchromosome-specific DNA is carried out using standard geneticengineering techniques, e.g., Davies et al., “Cloning of aRepresentative Genomic Library of the Human X Chromosome After Sortingby Flow Cytometry,” Nature, Vol. 293, pgs. 374-376 (1981); Krumlauf etal., “Construction and Characterization of Genomic Libraries fromSpecific Human Chromosomes,” Proc. Natl. Acad. Sci., Vol. 79, pgs.2971-2975 (1982); Lawn et al., “The Isolation and Characterization ofLinked Delta-and-Beta-Globin Genes from a Cloned Library of Human DNA.”Cell, Vol. 15, pgs. 1157-1174 (1978); and Maniatis et al., “MolecularCloning: A Laboratory Manual,” (Cold Springs Harbor Laboratory, 1982),pgs. 256-308; Van Dilla et al., id, Fuscoe, Gene, 52:291 (1987); andFuscoe et al., Cytogenet. Cell Genet., 43:79 (1986). Said references areherein incorporated by reference.

[0199] Recombinant DNA libraries for each of the human chromosomes havebeen constructed by the National Laboratory Gene Library Project and areavailable from the American Type Culture Collection. [Van Dilla et al.,Biotechnology, 4:537 (1986).] Small insert-containing libraries wereconstructed by complete digestion of flow sorted human chromosomegenomic DNA with HindIII or EcoRI and cloning into the Lambda insertionvector Charon 21A. The vector is capable of accepting human inserts ofup to 9.1′ kb in size. Thus, HindIII (or EcoRI) restriction fragmentsgreater than 9.1′ kb will not be recovered from these libraries. Theobserved average insert size in these libraries is approximately 4 kb. Arepresentative list of the HindIII chromosome-specific libraries withtheir ATCC accession numbers are shown in Table 1. TABLE 1 HUMANCHROMOSOME - SPECIFIC GENOMIC LIBRARIES IN CHARON 21A VECTOR CHROMOSOMEATCC # LIBRARY  1 57753 LL01NS01  1 57754 LL01NS02  2 57744 LL02NS01  357751 LL03NS01  4 57700 LL04NS01  4 57745 LL04NS02  5 57746 LL05NS01  657701 LL06NS01  7 57755 LL07NS01  8 57702 LL08NS02  9 57703 LL09NS01 1057736 LL10NS01 11 57704 LL11NS01 12 57756 LL12NS01 13 57705 LL13NS01 1357757 LL13NS02 14 57706 LL14NS01 14/15 57707 LL99NS01 15 57737 LL15NS0116 57758 LL16N503 17 57759 LL17NS02 18 57710 LL18NS01 19 57711 LL19NS0120 57712 LL20NS01 21 57713 LL21NS02 22 57714 LL22NS01 X 57747 LL0XNS01 Y57715 LL0YNS01

[0200] Alternatively, the extracted DNA from a sorted chromosome typecan be amplified by the polymerase chain reaction (PCR) rather thancloning the extracted DNA in a vector or propagating it in a cell line.Appropriate tails are added to the extracted DNA in preparation for PCR.References for such PCR procedures are set out in Section I.B infra.

[0201] Other possible methods of isolating the desired sequences fromhybrid cells include those of Schmeckpeper et al., “Partial Purificationand Characterization of DNA from Human X Chromosome,” Proc. Natl. Acad.Sci., Vol. 76, pgs. 6525-6528 (1979); or Olsen et al., supra (inBackground). Accordingly, these references are incorporated byreference.

[0202] 2. Isolating DNA from a Portion of a Chromosome. Among themethods that can be used for isolating region-specific chromosomal DNAinclude the selection of an appropriate chromosomal region from DNA thathas previously been mapped, for example, from a library of mappedcosmids; the sorting of derivative chromosomes, for example, by FACS;the microdissection of selected chromosomal material; subtractivehybridization; identification of an appropriate hybrid cell containing adesired chromosomal fragment, extracting and amplifying the DNA, andselecting the desired amplified DNA; and the selection of appropriatechromosomal material from radiation hybrids. The standard geneticengineering techniques outlined above in subsection I.A.1 are used insuch procedures well-known to those in the art. Amplification of theregion-specific DNA can be performed by cloning in an appropriatevector, propagating in an appropriate cell line, and/or by the use ofPCR (see I.B infra).

[0203] A preferred method of isolating chromosomal region-specific DNAis to use mapped short DNA sequences to probe a library of longer DNAsequences, wherein the latter library has usually been cloned in adifferent vector. For example, a probe cloned in a plasmid can be usedto probe a cosmid or yeast artificial chromosome (YAC) library. By usingan initial seed probe, overlapping clones in the larger insert librarycan be found (a process called “walking”), and a higher complexity probecan be produced for reliable staining of the chromosomal regionsurrounding the seed probe. Ultimately, when an entire genome for aspecies has been mapped (for example, by the Human Genome Project forthe human species), ordered clones for the entire genome of the specieswill be available. One can then easily select the appropriate clones toform a probe of the desired specificity.

[0204] Another method of isolating DNA from a chromosomal region orregions (or also a whole chromosome) is to propagate such a chromosomalregion or regions in an appropriate cell line (for example, a hybridcell line such as a human/hamster hybrid cell), extract the DNA from thecell line and clone it in an appropriate vector and select clonescontaining human DNA to form a library. When a hybrid cell is used, thechromosomes in the hybrid cell containing the human chromosomal materialmay be separated by flow sorting (FACS) prior to cloning to increase thefrequency of human clones in the library. Still further, total DNA fromthe hybrid cell can be isolated and labeled without further cloning andused as a probe, as exemplified in FIG. 6.

[0205] 3. Single-Stranded Probes. In some cases, it is preferable thatthe nucleic acid fragments of the heterogeneous mixture consist ofsingle-stranded RNA or DNA. Under some conditions, the bindingefficiency of single-stranded nucleic acid probes has been found to behigher during in situ hybridization, e.g., Cox et al., “Detection ofmRNAs in Sea Urchin Embryos by In Situ Hybridization Using AsymmetricRNA Probes,” Developmental Biology, Vol. 101, pgs. 485-502 (1984).

[0206] Standard methods are used to generate RNA fragments from isolatedDNA fragments. For example, a method developed by Green et al.,described Cell, Vol. 32, pgs. 681-694 (1983), is commercialy availablefrom Promega Biotec (Madison, Wis.) under the tradename “Riboprobe.”Other transcription kits suitable for use with the present invention areavailable from United States Biochemical Corporation (Cleveland, Ohio)under the tradename “Genescribe.” Single-stranded DNA probes can beproduced with the single-stranded bacteriophage M13, also available inkit form, e.g. Bethesda Research Labora-tories (Gaithersburg, Md.). Thehybridizations illustrated in FIG. 4 were performed with the librariesof Table 1 subcloned into the Bluescribe plasmid vector (Stratagene, LaJolla, Calif.). The Bluescribe plasmid contains RNA promoters whichpermit production of single-stranded probes.

[0207] Co-pending, commonly owned U.S. patent application Ser. No.934,188 (filed Nov. 24, 1986), entitled “Method of Preparing andApplying Single Stranded DNA Probes to Double Stranded Target DNAs,”provides methods for preparing and applying non-self-complementarysingle-stranded nucleic acid probes that improve signal-to-noise ratiosattainable in in situ hybridization by reducing non-specific andmismatched binding of the probe. That application further provides formethods of denaturing double-stranded target nucleic acid whichminimizes single-stranded regions available for hybridization that arenon-complementary to probe sequences. Said application is hereinspecifically incorporated by reference. Briefly, probe is constructed bytreating DNA with a restriction enzyme and an exonuclease to formtemplate/primers for a DNA polymerase. The digested strand isresynthesized in the presence of labeled nucleoside triphosphateprecursor, and the labeled single-stranded fragments are separated fromthe resynthesized fragments to form the probe. The target nucleic acidis treated with the same restriction enzyme used to construct the probe,and is treated with an exonuclease before application of the probe.

I.B. PCR

[0208] Another method of producing probes of this invention includes theuse of the polymerase chain reaction [PCR]. [For an explanation of themechanics of PCR, see Saiki et al., Science, 230:1350 (1985) and U.S.Pat. Nos. 4,683,195, 4,683,202 (both issued Jul. 28, 1987) and 4,800,159(issued Jan. 24, 1989).] Target-specific nucleic acid sequences,isolated as indicated above, can be amplified by PCR to producetarget-specific sequences which are reduced in or free of repetitivesequences. The PCR primers used for such a procedure are for the ends ofthe repetitive sequences, resulting in amplification of sequencesflanked by the repeats.

[0209]FIG. 7 illustrates such a method of using PCR wherein therepresentative repetitive sequence is Alu. If only short segments areamplified, it is probable that such sequences are free of other repeats,thus providing DNA reduced in repetitive sequences.

[0210] One can further suppress production of repetitive sequences insuch a PCR procedure by first hybridizing complementary sequences tosaid repetitive sequence wherein said complementary sequences haveextended non-complementary flanking ends or are terminated innucleotides which do not permit extension by the polymerase. Thenon-complementary ends of the blocking sequences prevent the blockingsequences from acting as a PCR primer during the PCR process.

II. REMOVAL OF REPETITIVE SEQUENCES AND/OR DISABLING THE HYBRIDIZATIONCAPACITY OF REPETITIVE SEQUENCES

[0211] Typically a probe of the current invention is produced in anumber of steps including: obtaining source nucleic acid sequences thatare complementary to the target region of the genome, labeling andotherwise processing them so that they will hybridize efficiently to thetarget and can be detected after they bind, and treating them to eitherdisable the hybridization capacity or remove a sufficient proportion ofshared repetitive sequences, or both disable and remove such sequences.The order of these steps depends on the specific procedures employed.

[0212] The following methods can be used to remove shared repetitivesequences and/or disable the hybridization capacity of such sharedrepetitive sequences. Such methods are representative and are expressedschematically in terms of procedures well known to those of ordinaryskill the art, and which can be modified and extended according toparameters and procedures well known to those in the art.

[0213] 1. Single-copy probes. A single-copy probe consists of nucleicacid fragments that are complementary to single-copy sequences containedin the target region of the genome. One method of constructing such aprobe is to start with a DNA library produced by cloning the targetregion. Some of the clones in the library will contain DNA whose entiresequence is single-copy; others will contain repetitive sequences; andstill others will have portions of single-copy and repetitive sequences.Selection, on a clone by done basis, and pooling of those clonescontaining only single-copy sequences will result in a probe that willhybridize specifically to the target region. The single-copy nature of aclone can ultimately be established by Southern hybridization usingstandard techniques. FIG. 4H shows hybridization with 120 clonesselected in this way from a chromosome 4 library.

[0214] Southern analysis is very time consuming and labor intensive.Therefore, less perfect but more efficient screening methods forobtaining candidate single-copy clones are useful. In Section V.B,examples of improved methods are provided for screening individual phageand plasmid clones for the presence of repetitive DNA usinghybridization with genomic DNA. The screening of plasmid clones is moreefficient, and approximately 80% of selected clones contain onlysingle-copy sequences; the remainder contain low-copy repeats. However,probes produced in this way can produce adequate staining contrast,indicating that the low-copy repetitive sequences can be tolerated inthe probe (see subsection 3 of this section).

[0215] A disadvantage of clone by clone procedures is that a clone isdiscarded even if only a portion of the sequence it contains isrepetitive. The larger the length of the cloned nucleic acid, thegreater the chance that it will contain a repetitive sequence.Therefore, when nucleic acid is propagated in a vector that containslarge inserts such as a cosmid, YAC, or in a cell line, such as hybridcells, it may be advantageous to subclone it in smaller pieces beforethe single-copy selection is performed. The selection procedures justoutlined above do not discriminate between shared and specificrepetitive sequences; clones with detectable repetitive sequences ofeither type are not used in the probe.

[0216] 2. Individual testing of hybridization properties. Thehybridization specificity of a piece of nucleic acid, for example, adone, can be tested by in situ hybridization. If under appropriatehybridization conditions it binds to single-copy or repetitive sequencesspecific for the desired target region, it can be included in the probe.Many sequences with specific hybridization characteristics are alreadyknown, such as chromosome-specific repetitive sequences [Trask et al.,supra, (1988) and references therein], VNTRs, numerous mapped singlecopy sequences. More are continuously being mapped. Such sequences canbe included in a probe of this invention.

[0217] 3. Bulk Procedures. In many genomes, such as the human genome, amajor portion of shared repetitive DNA is contained in a few families ofhighly repeated sequences such as Alu. A probe that is substantiallyfree of such high-copy repetitive sequences will produce useful stainingcontrast in many applications. Such a probe can be produced from somesource of nucleic acid sequences, for example, the libraries of Table I,with relatively simple bulk procedures. Therefore, such bulk proceduresare the preferred methods for such applications.

[0218] These methods primarily exploit the fact that the hybridizationrate of complementary nucleic acid strands increases as theirconcentration increases. Thus, if a heterogeneous mixture of nucleicacid fragments is denatured and incubated under conditions that permithybridization, the sequences present at high concentration will becomedouble-stranded more rapidly than the others. The double-strandednucleic add can then be removed and the remainder used as a probe.Alternatively, the partially hybridized mixture can be used as theprobe, the double-stranded sequences being unable to bind to the target.The following are methods representative of bulk procedures that areuseful for producing the target-specific staining of this invention.

[0219] 3a. Self-reassociation of the probe. Double-stranded probenucleic acid in the hybridization mixture is denatured and thenincubated under hybridization conditions for a time sufficient for thehigh-copy sequences in the probe to become substantiallydouble-stranded. The hybridization mixture is then applied to thesample. The remaining labeled single-stranded copies of the highlyrepeated sequences bind throughout the sample producing a weak, widelydistributed signal. The binding of the multiplicity of low-copysequences specific for the target region of the genome produce an easilydistinguishable specific signal.

[0220] Such a method is exemplified in Section VI.B (infra) withchromosome-specific libraries for chromosomes 4 and 21 (pBS4 and pBS21)as probes for those chromosomes. The hybridization mix, containing aprobe concentration in the range of 1-10 ng/ul was heated to denaturethe probe and incubated at 37° C. for 24 hours prior to application tothe sample.

[0221] 3b. Use of blocking nucleic acid. Unlabeled nucleic acidsequences which are complementary to those sequences in the probe whosehybridization capacity it is desired to inhibit are added to thehybridization mixture. The probe and blocking nucleic acid aredenatured, if necessary, and incubated under appropriate hybridizationconditions. The sequences to be blocked become double-stranded morerapidly than the others, and therefore are unable to bind to the targetwhen the hybridization mixture is applied to the target. In some cases,the blocking reaction occurs so quickly that the incubation period canbe very short, and adequate results can be obtained if the hybridizationmix is applied to the target immediately after denaturation. A blockingmethod is generally described by Sealy et al., “Removal of RepeatSequences form Hybridization Probes”, Nucleic Acid Research 13:1905(1985), which reference is incorporated by reference. Examples ofblocking nucleic acids include genomic DNA, a high-copy fraction ofgenomic DNA and particular sequences as outlined below (i-iii).

[0222] 3b.i. Genomic DNA. Genomic DNA contains all of the nucleic acidsequences of the organism in proportion to their copy-number in thegenome. Thus, adding genomic DNA to the hybridization mixture increasesthe concentration of the high-copy repeat sequences more than low-copysequences, and therefore is more effective at blocking the former.However, the genomic DNA does contain copies of the sequences that arespecific to the target and so will also reduce the desiredchromosome-specific binding if too much is added. Guidelines todetermine how much genomic DNA to add (see 3.e. Concept of Q, infra) andexamples of using genomic blocking DNA are provided below. The blockingeffectiveness of genomic DNA can be enhanced under some conditions byadjusting the timing of its addition to the hybridization mix; examplesof such timing adjustments are provided with Protocol I and Protocol IIhybridizations illustrated in FIGS. 4B through E (Protocol I) and FIG.4F (Protocol II) and detailed in Section VI, infra.

[0223] 3b.ii. High-copy fraction of genomic DNA. The difficulty with useof genomic DNA is that it also blocks the hybridization of the low-copysequences, which are predominantly the sequences that give the desiredtarget staining. Thus, fractionating the genomic DNA to obtain only thehigh-copy sequences and using them for blocking overcomes thisdifficulty. Such fractionation can be done, for example, withhydroxyapatite as described below (3c.i).

[0224] 3b.iii. Specified sequences. The blocking of a particularsequence in the probe can be accomplished by adding many unlabeledcopies of that sequence. For example, Alu sequences in the probe can beblocked by adding cloned Alu DNA. Blocking DNA made from a mixture of afew dones containing the highest copy sequences in the human genome canbe used effectively with chromosome-specific libraries for example,those of Table I. Alternatively, unlabeled nucleic acid sequences fromone or more chromosome-specific libraries could be used to block a probecontaining labeled sequences from one or more other chromosome-specificlibraries. The shared sequences would be blocked whereas sequencesoccurring only on the target chromosome would be unaffected. FIG. 4Fshows that genomic DNA was not effective in completely blocking thehybridization of a sequence or sequences shared by human chromosome 21and the centromeric regions of the other human acrocentric chromosomes.When a clone or clones containing such a sequence or sequences is or areeventually isolated, unlabeled DNA produced therefrom could be added tothe genomic blocking DNA to improve the specificity of the staining.

3c. REMOVAL OF SEQUENCES

[0225] 3c.i. Hydroxyapatite. Single- and double-stranded nucleic acidshave different binding characteristics to hydroxyapatite. Suchcharacteristics provide a basis commonly used for fractionating nucleicacids. Hydroxyapatite is commerically available (eg. Bio-RadLaboratories, Richmond, Calif.). The fraction of genomic DNA containingsequences with a particular degree of repetition, from the highestcopy-number to single-copy, can be obtained by denaturing genomic DNA,allowing it to reassociate under appropriate conditions to a particularvalue of C_(O)t, followed by separation using hydroxyapatite. Thesingle- and double-stranded nucleic acid can also be discriminated byuse of S1 nuclease. Such techniques and the concept of C_(O)t areexplained in Britten et al., “Analysis of Repeating DNA Sequences byReassociation”, in Methods in Enzymology Vol. 29, pgs. 363-418 (1974),which article is herein incorporated by reference.

[0226] The single-stranded nucleic acid fraction produced in 3a. or 3b.above can be separated by hydroxyapatite and used as a probe. Thus, thesequences that have been blocked (that become double-stranded) arephysically removed. The probe can then be stored until needed. The probecan then be used without additional blocking nucleic acid, or itsstaining contrast can perhaps be improved by additonal blocking.

[0227] 3c.ii. Reaction with immobilized nucleic acid. Removal ofparticular sequences can also be accomplished by attachingsingle-stranded “absorbing” nucleic add sequences to a solid supportSingle-stranded source nucleic acid is hybridized to the immobilizednucleic add. After the hybridization, the unbound sequences arecollected and used as the probe. For example, human genomic DNA can beused to absorb repetitive sequences from human probes. One such methodis described by Brison et al., “General Method for Cloning Amplified DNAby Differential Screening with Genomic Probes,” Molecular and CellularBiology, Vol. 2, pgs. 578-587 (1982). Accordingly, that reference isincorporated by reference. Briefly, minimally sheared human genomic DNAis bound to diazonium cellulose or a like support. The source DNA,appropriately cut into fragments, is hybridized against the immobilizedDNA to C_(O)t values in the range of about 1 to 100. The preferredstringency of the hybridization conditions may vary depending on thebase composition of the DNA. Such a procedure could remove repetitivesequences from chromosome-specific libraries, for example, those ofTable I, to produce a probe capable of staining a whole humanchromosome.

[0228] 3d. Blocking non-targeted sequences in the targeted genome.Blocking of non-targeted binding sites in the targeted genome byhybridization with unlabeled complementary sequences will preventbinding of labeled sequences in the probe that have the potential tobind to those sites. For example, hybridization with unlabeled genomicDNA will render the high-copy repetitive sequences in the target genomedoublestranded. Labeled copies of such sequences in the probe will notbe able to bind when the probe is subsequently applied.

[0229] In practice, several mechanisms combine to produce the stainingcontrast. For example, when blocking DNA is added to the probe as in 3babove, that which remains single-stranded when the probe is applied tothe target can bind to and block the target sequences. If the incubationof the probe with the blocking DNA is minimal, then the genomic DNAsimultaneously blocks the probe and competes with the probe for bindingsites in the target.

[0230] 3e. Concept of Q. As mentioned in section 3b.i above, it isnecessary to add the correct amount of genomic DNA to achieve the bestcompromise between inhibiting the hybridization capacity of high-copyrepeats in the probe and reducing the desired signal intensity byinhibition of the binding of the target-specific sequences. Thefollowing discussion pertains to use of genomic blocking DNA with probesproduced by cloning or otherwise replicating stretches of DNA from thetarget region of the genome. Thus, the probe contains a representativesampling of the single-copy, chromosome-specific repetitive sequences,and shared repetitive sequences found in the target. Such a probe mightrange in complexity from 100 kb of sequence derived from a small regionof the genome, for example several closely spaced cosmid clones; to manymillions of bases, for example a combination of multiple libraries fromTable I. The discussion below is illustrative and can be extended toother situations where different blocking nucleic acids are used. Thefollowing discussion of Q is designed only to give general guidelines asto how to proceed.

[0231] The addition of unlabeled genomic DNA to a hybridization mixcontaining labeled probe sequences increases the concentration of all ofthe sequences, but increases the concentration of the shared sequencesby a larger factor than the concentration of the target-specificsequences because the shared sequences are found elsewhere in thegenome, whereas the target-specific sequences are not. Thus, thereassociation of the shared sequences is preferentially enhanced so thatthe hybridization of the labeled copies of the shared sequences to thetarget is preferentially inhibited.

[0232] To quantity this concept, first consider one of the sequences,repeat or single-copy, that hybridize specifically to the ith chromosomein a hybridization mixture containing a mass m_(p) of probe DNA from theith chromosome library of Table 1 (for example) and m_(b) of unlabeledgenomic DNA. The number of labeled copies of the sequence isproportional to m_(p). However, the number of unlabeled copies isproportional to f_(i)m_(b), where f_(i) is the fraction of genomic DNAcontained on the ith chromosome. Thus, the ratio of unlabeled to labeledcopies of each of the sequences specific for the target chromosome, isf_(i)m_(b)/m_(p), which is defined herein as Q. For normal humanchromosomes, 0.016≦f_(i)≦0.08 [Mendelsohn et al., Science, 179:1126(1973)]. For representative examples described in Section VI.B (infra),f₄=0.066 and f₂₁=0.016. For a probe targeted at a region comprised of Lbase pairs, f_(i)=L/G where G is the number of base pairs in a genome(approximately 3×10⁹ bases for humans and other mammals). Thus, Q=(L/G)(m_(b)/m_(p)).

[0233] Now consider a shared sequence that is distributed more-or-lessuniformly over the genome, for example, Alu. The number of labeledcopies is proportional to m_(p), whereas the number of unlabeled copiesis proportional to m_(b). Thus, the ratio of unlabeled to labeled copiesis m_(b)/m_(p)=Q/f_(i). This is true for all uniformly distributedsequences, regardless of copy number. Thus adding genomic DNA increasesthe concentration of each specific sequence by the factor 1+Q, whereaseach uniformly distributed sequence is increased by the larger factor1+Q/f_(i). Thus, the reassociation rates of the shared sequences areincreased by a larger factor than those of the specific sequences by theaddition of genomic DNA.

[0234] It can be shown that roughly half of the beneficial effect ofgenomic DNA on relative reassociation rates is achieved when Q=1, and,by Q=5, there is essentially no more benefit to be gained by furtherincreases. Thus, the protocol I hybridizations of Section VI.B infrakeep Q≦5.

[0235] To illustrate the use of genomic blocking DNA, it is convenientto consider a model of a genome wherein 50% of the DNA is comprised ofspecific sequences (both repetitive and single-copy) and the other 50%of the DNA is comprised of shared repetitive sequences that aredistributed uniformly over the genome. Thus, according to the model, ifthe target is L bases (that is, the probe contains fragmentsrepresenting L bases of the target area or areas of the genome),sequences containing L/2 bases will be specific to the target, and L/2will be shared with the entire genome.

[0236] Case I. The complexity of the probe is about 50 kb to about 100kb. (In this case the complexity may be approximately equal to L sincethe probability is that no repetitive sequences will typically occurwith more than a few copies in such a number of bases). Using a standardhybridization mixture (as exemplified in Section VI.B, infra), thetarget can be hybridized with about 2 ng of labeled probe DNA in 10 ulof hybridization mix, corresponding to approximately 1 pg/ul per kb ofspecific sequences (as used in Section VI.B, infra). Suppose thehybridization is to a slide containing 10⁴⁴ cells (a typical number),and each cell has about 6 pg of DNA, (typical for mammals). Then in thismodel calculation, there is 3 pg of shared repetitive sequences percell. Thus, for 10⁴ cells there are 3×10⁴ pg or 30 ng of sharedsequences on the slide. Similarly, there is 10⁴×0.5×10⁵×6/3×10⁹ pg=1 pgof target for the specific sequences. The probe contains ½×2 ng or 1 ngof shared sequences and 1 ng of specific sequences. Therefore, there isnot enough probe to saturate the shared sequences in the target DNA, butenough to saturate the specific sequences. The signal from the sharedsequences is spread at low intensity over the entire genome whereas thespecific signal is concentrated in a compact region. Thus, good contrastcan be obtained without adding any blocking genomic DNA at all.

[0237] A great deal of genomic DNA can be added to improve the contrastwithout interfering with the hybridization of the specific sequences,that is, Q remains low even if a great deal of genomic DNA is added.

Q=10⁵/3×10⁹ m _(b) /m _(p)=3×10⁻⁵ m _(b) /m _(p)

[0238] If a large amount of blocking nucleic acid, for example, 10 ugwere used (according to the standard hybridization protocols exemplifiedin Section VI.B infra wherein the practical limit of total nucleic acidis on the order of 10 ug in a 10′ ul hybridization mixture) with the 2ng of probe, then Q=3×10⁻⁵×10⁴ ng/2 ng=3/2×10⁻¹=0.15. Thus, Q is <1, andthe blocking DNA cannot substantially interfere with the desired signal.Increasing the amount of labeled probe nucleic acid to speed thehybridization would further decrease Q. In practice, one would typicallyuse 1 ug of blocking DNA for such a hybridization.

[0239] Case II. As the size of the target region is increased, thecomplexity of the probe necessarily is increased, and the amount of DNAin the hybridization mix needs to be increased in order to have asufficient concentration of each portion of specific sequence tohybridize. Also, if one desires to decrease the hybridization time ofthe procedure, the probe concentration must be increased. In thesesituations, the increase in probe concentration results in an increasein the amount of shared sequences in the hybridization mixture, which inturn increases the amount of hybridization that will occur to the sharedsequences in the target area or areas, thereby reducing the contrastratio.

[0240] With very high complexity probes spanning several entirechromosomes, L/G can approach 1. In order to stain such a portion of thegenome within a reasonable time, for example, overnight, theconcentration of labeled nucleic acid needs to be increased, forexample, 200 ng in 10 ul of hybridization mixture. Up to about 3000 ngof blocking DNA can be used and still keep Q≦5[wherein the calculationis Q=5=0.3 m_(b)/200 n_(g) or m_(b)=1000 ng/0.3=3,333 ng]. In practice,staining 25% and more of the human genome (for example, humanchromosomes 1, 3 and 4) can be accomplished with the blocking protocolsdescribed below, but the contrast is less than for that achieved withprobes for smaller regions.

III. LABELING THE NUCLEIC ACID FRAGMENTS OF THE HETEROGENEOUS MIXTURE

[0241] Several techniques are available for labeling single- anddouble-stranded nucleic acid fragments of the heterogeneous mixture.They include incorporation of radioactive labels, e.g. Harper et al.Chromosoma, Vol 83, pgs. 431-439 (1984); direct attachment offluorochromes or enzymes, e.g. Smith et al., Nucleic Acids Research,Vol. 13, pgs. 2399-2412 (1985), and Connolly et al., Nucleic AcidsResearch, Vol. 13, pgs. 4485-4502 (1985); and various chemicalmodifications of the nucleic acid fragments that render them detectableimmunochemically or by other affinity reactions, e.g. Tchen et al.,“Chemically Modified Nucleic Acids as Immunodetectable Probes inHybridization Experiments,” Proc. Natl. Acad. Sci., Vol 81, pgs.3466-3470 (1984); Richardson et al., “Biotin and Fluorescent Labeling ofRNA Using T4 RNA Ligase,” Nucleic Acids Research, Vol. 11, pgs.6167-6184 (1983); Langer et al., “Enzymatic Synthesis of Biotin-LabeledPolynucleotides: Novel Nucleic Acid Affinity Probes,” Proc. Natl. Acad.Sci., Vol. 78, pgs. 6633-6637 (1981); Brigati et al., “Detection ofViral Genomes in Cultured Cells and Paraffin-Embedded Tissue SectionsUsing Biotin-Labeled Hybridization Probes,” Virology, Vol. 126, pgs.32-50 (1983); Broker et al., “Electron Microscopic Visualization of tRNAGenes with Ferritin-Avidin: Biotin Labels,” Nucleic Acids Research, Vol.5, pgs. 363-384 (1978); Bayer et al., “The Use of the Avidin BiotinComplex as a Tool in Molecular Biology,” Methods of BiochemicalAnalysis, Vol. 26, pgs. 1-45 (1980) Kuhlmann, Immunoenzyme Techniques inCytochemistry (Weinheim, Basel, 1984). Langer-Safer et al., PNAS USA,79:4381 (1982): Landegent et al., Exp. Cell Res., 153:61 (1984); andHopman et al., Exp. Cell Res., 169:357 (1987).

[0242] Exemplary labeling means include those wherein the probefragments are biotinylated, modified withN-acetoxy-N-2-acetylaminofluorene, modified with fluoresceinisothiocyanate, modified with mercury/TNP ligand, sulfonated,digoxigenenated or contain T-T dimers.

[0243] The key feature of “probe labeling” is that the probe bound tothe target be detectable. In some cases, an intrinsic feature of theprobe nucleic acid, rather than an added feature, can be exploited forthis purpose. For example, antibodies that specifically recognizeRNA/DNA duplexes have been demonstrated to have the ability to recognizeprobes made from RNA that are bound to DNA targets [Rudkin and Stollar,Nature, 265:472-473(1977)]. The RNA used for such probes is unmodified.Probe nucleic acid fragments can be extended by adding “tails” ofmodified nucleotides or particular normal nucleotides. When a normalnucleotide tail is used, a second hybridization with nucleic acidcomplementary to the tail and containing fluorochromes, enzymes,radioactivity, modified bases, among other labeling means, allowsdetection of the bound probe. Such a system is commerically availablefrom Enzo Biochem (Biobridge Labeling System; Enzo Biochem Inc., NewYork, N.Y.).

[0244] Another example of a means to visualize the bound probe whereinthe nucleic acid sequences in the probe do not directly carry somemodified constituent is the use of antibodies to thymidine dimers.Nakane et al., 20 (2):229 (1987), illustrate such a method whereinthymine-thymine dimerized DNA (T-T DNA) was used as a marker for in situhybridization. The hybridized T-T DNA was detected immunohistochemicallyusing rabbit anti-T-T DNA antibody.

[0245] All of the labeling techniques disclosed in the above referencesmay be preferred under particular circum-stances. Accordingly, theabove-cited references are in-corporated by reference. Further, anylabeling techniques known to those in the art would be useful to labelthe staining compositions of this invention. Several factors govern thechoice of labeling means, including the effect of the label on the rateof hybridization and binding of the nucleic acid fragments to thechromosomal DNA, the accessibility of the bound probe to labelingmoieties applied after initial hybridization, the mutual compatibilityof the labeling moieties, the nature and intensity of the signalgenerated by the label, the expense and ease in which the label isapplied, and the like.

[0246] Several different high complexity probes, each labeled by adifferent method, can be used simultaneously. The binding of differentprobes can thereby be distinguished, for example, by different colors.

IV. IN SITU HYBRIDIZATION

[0247] Application of the heterogeneous mixture of the invention tochromosomes is accomplished by standard in situ hybridizationtechniques. Several excellent guides to the technique are available,e.g., Gall and Pardue, “Nucleic Acid Hybridization in CytologicalPreparations,” Methods in Enzymology, Vol. 21, pgs. 470-480 (1981);Henderson, “Cytological Hybridization to Mammalian Chromo-somes,”International Review of Cytology, Vol. 76, pgs. 146 (1982); and Angerer,et al., “In Situ Hybridization to Cellular RNAs,” in GeneticEngineering: Principles and Methods, Setlow and Hollaender, Eds., Vol.7, pgs. 43-65 (Plenum Press, New York, 1985). Accordingly, thesereferences are incorporated by references.

[0248] Three factors influence the staining sensitivity of thehybridization probes: (1) efficiency of hybridization (fraction oftarget DNA that can be hybridized by probe), (2) detection efficiency(i.e., the amount of visible signal that can be obtained from a givenamount of hybridization probe), and (3) level of noise produced bynonspecific binding of probe or components of the detection system.

[0249] Generally in situ hybridization comprises the following majorsteps: (1) fixation of tissue or biological structure to be examined,(2) prehybridization treatment of the biological structure to increaseaccessibility of target DNA, and to reduce nonspecific binding, (3)hybridization of the heterogeneous mixture of probe to the DNA in thebiological structure or tissue; (4) posthybridization washes to removeprobe not bound in specific hybrids, and (5) detection of the hybridizedprobes of the heterogeneous mixture. The reagents used in each of thesesteps and their conditions of use vary depending on the particularsituation.

[0250] The following comments are meant to serve as a guide for applyingthe general steps listed above. Some experimentation may be required toestablish optimal staining conditions for particular applications.

[0251] In preparation for the hybridization, the probe, regardless ofthe method of its production, may be broken into fragments of the sizeappropriate to obtain the best intensity and specificity ofhybridization. As a general guideline concerning the size of thefragments, one needs to recognize that if the fragments are too longthey are not able to penetrate into the target for binding and insteadform aggregates that contribute background noise to the hybridization;however, if the fragments are too short, the signal intensity isreduced.

[0252] Under the conditions of hybridization exemplified in Section VI.Bwherein human genomic DNA is used as an agent to block the hybridizationcapacity of the high copy shared repetitive sequences, the preferredsize range of the probe fragments is from about 200 bases to about 2000bases, more preferably in the vicinity of 1 kb. When the size of theprobe fragments is in about the 800 to about 1000 base range, thepreferred hybridization temperature is about 30° C. to about 45° C.,more preferably about 35° C. to about 40° C., and still more preferablyabout 37° C.; preferred washing temperature range is from about 40° C.to about 50° C., more preferably about 45° C.

[0253] The size of the probe fragments is checked before hybridizationto the target; preferably the size of the fragments is monitored byelectrophoresis, more preferably by denaturing agarose gelelectrophoresis.

[0254] Fixatives include acid alcohol solutions, acid acetone solutions,Petrunkewitsch's reagent, and various aldehydes such as formaldehyde,paraformaldehyde, glutaraldehyde, or the like. Preferably,ethanol-acetic acid or methanol-acetic acid solutions in about 3:1proportions are used to fix the chromosomes in metaphase spreads. Forcells or chromosomes in suspension, a fixation procedure disclosed byTrask, et al., in Science, Vol. 230, pgs. 1401-1402 (1985), is useful.Accordingly, Trask et al., is incorporated by reference. Briefly, K₂CO₃and dimethylsuberimidate (DMS) are added (from a 5× concentrated stocksolution, mixed immediately before use) to a suspension containing about5×10⁶ nuclei/ml. Final K₂CO₃ and DMS concentrations are 20 mM and 3 mM,respectively. After 15 minutes at 25° C., the pH is adjusted from 10.0to 8.0 by the addition of 50 microliters of 100 mM citric acid permilliliter of suspension. Nuclei are washed once by centrifugation (300g, 10 minutes, 4° C. in 50 mM kCl, 5 mM Hepes buffer, at pH 9.0, and 10mM MgSO₄).

[0255] A preferred fixation procedure for cells or nuclei in suspensionis disclosed by Trask et al., Hum. Genet. 78:251-259 (1988), whicharticle is herein incorporated by reference. Briefly, nuclei are fixedfor about 10 minutes at room temperature in 1% paraformaldehyde in PBS,50′ mM MgSO₄, pH 7.6 and washed twice. Nuclei are resuspended inisolation buffer (IB) (50 mM KC1, 5 mM HEPES, 10 mM MgSO₄, 3 mMdithioerythritol, 0.15 mg/ml RNase, pH 8.0)/0.05% Triton X-100 at10⁸/ml.

[0256] Frequently before in situ hybridization chromosomes are treatedwith agents to remove proteins. Such agents include enzymes or mildadds. Pronase, pepsin or proteinase K are frequently used enzymes. Arepresentative acid treatment is 0.02-0.2 N HC1, followed by hightemperature (e.g., 70° C.) washes. Optimization of deproteinizationrequires a combination of protease concentration and digestion time thatmaximizes hybridization, but does not cause unacceptable loss ofmorphological detail. Optimum conditions vary according to tissue typesand method of fixation. Additional fixation after protease treatment maybe useful. Thus, for particular applications, some experimentation maybe required to optimize protease treatment.

[0257] In some cases pretreatment with RNase may be desirable to removeresidual RNA from the target. Such removal can be accomplished byincubation of the fixed chromosomes in 50-100 microgram/milliliter RNasein 2× SSC (where SSC is a solution of 0.15M NaCL and 0.015M sodiumcitrate) for a period of 1-2 hours at room temperature.

[0258] The step of hybridizing the probes of the heterogeneous probemixture to the chromosomal DNA involves (1) denaturing the target DNA sothat probes can gain access to complementary single-stranded regions,and (2) applying the heterogeneous mixture under conditions which allowthe probes to anneal to complementary sites in the target. Methods fordenaturation include incubation in the presence of high pH, low pH, hightemperature, or organic solvents such as formamide, tetraalkylammoniumhalides, or the like, at various combinations of concentration andtemperature. Single-stranded DNA in the target can also be produced withenzymes, such as, Exonudease III [van Dekken et al., Chromosoma (Berl)97:1-5 (1988)]. The preferred denaturing procedure is incubation forbetween about 1-10 minutes in formamide at a concentration between about35-95 percent in 2× SSC and at a temperature between about 25-70° C.Determination of the optimal incubation time, concentration, andtemperature within these ranges depends on several variables, includingthe method of fixation and type of probe nucleic acid (for example, DNAor RNA).

[0259] After the chromosomal DNA is denatured, the denaturing agents aretypically removed before application of the heterogeneous probe mixture.Where formamide and heat are the primary denaturing agents, removal isconveniently accomplished by several washes with a solvent, whichsolvent is frequently chilled, such as a 70%, 85%, 100% cold ethanolseries. Alternatively the composition of the denaturant can be adjustedas appropriate for the in situ hybridization by addition of otherconsitutents or washes in appropriate solutions. The probe and targetnucleic acid may be denatured simultaneously by applying thehybridization mixture and then heating to the appropriate temperature.

[0260] The ambient physiochemical conditions of the chromosomal DNA andprobe during the time the heterogeneous mixture is applied is referredto herein as the hybridization conditions, or annealing conditions.Optimal hybridization conditions for particular applications can beadjusted by controlling several factors, including concentration of theconstituents, incubation time of chromosomes in the heterogeneousmixture, and the concentrations, complexities, and lengths of thenucleic acid fragments making up the heterogeneous mixture. Roughly, thehybridization conditions must be sufficiently close to the meltingtemperature to minimize nonspecific binding. On the other hand, theconditions cannot be so stringent as to reduce correct hybridizations ofcomplementary sequences below detectable levels or to requireexcessively long incubation times.

[0261] The concentrations of nucleic acid in the hybridization mixtureis an important variable. The concentrations must be high enough so thatsufficient hybridization of respective chromosomal binding sites occursin a reasonable time (e.g., within hours to several days). Higherconcentrations than that necessary to achieve adequate signals should beavoided so that nonspecific binding is minimized. An important practicalconstraint on the concentration of nucleic acid in the probe in theheterogeneous mixture is solubility. Upper bounds exist with respect tothe fragment concentration, i.e., unit length of nucleic add per unitvolume, that can be maintained in solution and hybridize effectively.

[0262] In the representational examples described in Section VI.B(infra), the total DNA concentration in the hybridization mixture had anupper limit on the order of 1 ug/ul. Probe concentrations in the rangeof 1-20 ng/ul were used for such whole chromosome staining. The amountof genomic blocking DNA was adjusted such that Q was less than 5. At thelow end of probe concentration, adequate signals were obtained with aone hour incubation, that is, a time period wherein the probe andblocking DNA are maintained together before application to the targetedmaterial, to block the high-copy sequences and a 16 hour hybridization.Signals were visible after two hours of hybridization. The best results(bright signals with highest contrast) occurred after a 100 hourhybridization, which gave the low-copy target-specific sequences moreopportunity to find binding sites. At the high end of the probeconcentration, bright signals are obtained after hybridizations of 16hours or less; the contrast was reduced since more labeled repetitivesequences were included in the probe.

[0263] The fixed target object can be treated in several ways eitherduring or after the hybridization step to reduce nonspecific binding ofprobe DNA.

[0264] Such treatments include adding nonprobe, or “carrier”, DNA to theheterogeneous mixture, using coating solutions, such as Denhardt'ssolution (Biochem. Biophys. Res. Commun., Vol. 23, pgs. 641-645 (1966),with the heterogeneous mixture, incubating for several minutes, e.g.,5-20, in denaturing solvents at a temperature 5-10° C. above thehybridization temperature, and in the case of RNA probes, mild treatmentwith single strand RNase (e.g., 5-10 micrograms per millileter RNase) in2× SSC at room temperature for 1 hour).

V. CHROMOSOME-SPECIFIC STAINING REAGENTS COMPRISING SELECTED SINGLE-COPYSEQUENCES V.A. MAKING AND USING A STAINING REAGENT SPECIFIC TO HUMANCHROMOSOME 21 V.A.1. ISOLATION OF CHROMOSOME 21 and CONSTRUCTION OF ACHROMOSOME 21-SPECIFIC LIBRARY

[0265] DNA fragments from human chromosome-specific libraries areavailable from the National Laboratory Gene Library Project through theAmerican Type Culture Collection (ATCC), Rockville, Md. DNA fragmentsfrom chromosome 21 were generated by the procedure described by Fuscoeet al., in “Construction of Fifteen Human Chromosome-Specific DNALibraries from Flow-Purified Chromosomes,” Cytogenet. Cell Genet., Vol.43, pgs. 79-86 (1986), which reference is incorporated by reference.Briefly, a human diploid fibroblast culture was established from newbornforeskin tissue. Chromosomes of the cells were isolated by the MgSO₄method of van den Engh et al., Cytometery, Vol. 5, pgs. 108-123 (1984),and stained with the fluorescent dyes—Hoechst 33258 and Chromomycin A3.Chromsome 21 was purified on the Lawrence Livermore National Laboratoryhigh speed sorter, described by Peters et al., Cytometry, Vol. 6, pgs.290-301 (1985).

[0266] After sorting, chromosome concentrations were approximately4×10⁵/ml. Therefore, prior to DNA extraction, the chromosomes(0.2-1.0×10 ⁶) were concen-trated by centrifugation at 40,000×g for 30minutes at 4° C. The pellet was then resuspended in 100 microliters ofDNA isolation buffer (15 mM NaCl, 10 mM EDTA, 10 mM Tris HCl pH 8.0)containing 0.5% SDS and 100 micrograms/ml proteinase K. After overnightincubation at 37° C., the proteins were extracted twice withphenol:chloroform: isoamyl alcohol (25:24:1) and once withchloroform:isoamyl alcohol (24:1). Because of the small amounts of DNA,each organic phase was reextracted with a small amount of 10 mM Tris pH8.0, 1 mM EDTA (TE). Aqueous layers were combined and transferred to aSchleicher and Schuell mini-collodion membrane (#UHO20/25) and dialyzedat room temperature against TE for 6-8 hours. The purified DNA solutionwas then digested with 50 units of HindIII (Bethesda ResearchLaboratories, Inc.) in 50 mM NaCl, 10 mM Tris HCl pH 7.5, 10 mM MgCl₂, 1mM dithiothreitol. After 4 hours at 37° C., the reaction was stopped byextractions with phenol and chloroform as described above. The aqueousphase was dialyzed against water overnight at 4° C. in a mini-collodionbag and then 2 micrograms of Charon 21A arms cleaved with HindIII andtreated with calf alkaline phosphatase (Boehringer Mannheim) were added.This solution was concentrated under vacuum to a volume of 50-100microliters and transferred to a 0.5 ml microfuge tube where the DNA wasprecipitated with one-tenth volume 3M sodium acetate pH 5.0 and 2volumes ethanol. The precipitate was collected by centrifugation, washedwith cold 70% ethanol, and dissolved in 10 microliters of TE.

[0267] After allowing several hours for the DNA to dissolve, 1microliter of 10× ligase buffer (0.5M Tris HCl pH 7.4, 0.1 M MgCl₂, 0.1Mdithiothreitol, 10 mM ATP, 1 mg/ml bovine serum albumin) and 1 unit ofT4 ligase (Bethesda Research Laboratory, Inc.) were added. The ligationreaction was incubated at 10° C. for 16-20 hours and 3 microliteraliquots were packaged into phage particles using in vitro extractsprepared from E. coli strains BHB 2688 and BHB 2690, described by Hohnin Methods in Enzymology, Vol. 68, pgs. 299-309 (1979) MolecularCloning: A Laboratory Manual, (Cold Spring Harbor Laboratory, New York,1982). Briefly, both extracts were prepared by sonication and combinedat the time of in vivo packaging. These extracts packaged wild-typelambda DNA at an efficiency of 1-5×10 ⁸ plaque forming units (pfu) permicrogram. The resultant phage were amplified on E. coli LE392 at adensity of approximately 10⁴ pfu/150 mm dish for 8 hours to preventplaques from growing together and to minimize differences in growthrates of different recombinants. The phage were eluted from the agar in10 ml SM buffer (50 mM Tris HCl pH 7.5, 10 mM MgSO₄, 100 mM NaCl, 0.01%gelatin) per plate by gentle shaking at 4° C. for 12 hours. The plateswere then rinsed with an additional 4 ml of SM. After pelleting cellulardebris, the phage suspension was stored over chloroform at 4° C.

V.A.2. CONSTRUCTION AND USE OF CHROMOSOME 21-SPECIFIC STAIN FOR STAININGCHROMOSOME 21 OF HUMAN LYMPHOCYTES

[0268] Clones having unique sequence inserts are isolated by the methodof Benton and Davis, Science, Vol. 196, pgs. 180-182 (1977). Briefly,about 1000 recombinant phage are isolated at random from the chromosome21-specific library. These are transferred to nitrocellulose and probedwith nick translated total genomic human DNA.

[0269] Of the clones which do not show strong hybridization,approximately 300 are picked which contain apparent unique sequence DNA.After the selected clones are amplified, the chromosome 21 insert ineach clone is ³²P labeled and hybridized to Southern blots of humangenomic DNA digested with the same enzyme used to construct thechromosome 21 library, i.e., Hind III. Unique sequence containing clonesare recognized as those that produce a single band during Southernanalysis. Roughly, 100 such clones are selected for the heterogeneousmixture. The unique sequence clones are amplified, the inserts areremoved by Hind III digestions, and the inserts are separated from thephage arms by gel electrophoresis. The probe DNA fragments (i.e., theunique sequence inserts) are removed from the gel and biotinylated bynick translation (e.g., by a kit available from Bethesda ResearchLaboratories). Labeled DNA fragments are separated from the nicktranslation reaction using small spin columns made in 0.5 ml Eppendorphtubes filled with Sephadex G-50 (medium) swollen in 50 mM Tris, 1 mMEDTA, 0.1% SDS, at pH 7.5. Human lymphocyte chromosomes are preparedfollowing Harper et al, Proc. Natl. Acad. Sci., Vol. 78, pgs. 4458-4460(1981). Metaphase and interphase cells were washed 3 times in phosphatebuffered saline, fixed in methanol-acetic acid (3:1) and dropped ontocleaned microscope slides. Slides are stored in a nitrogen atmosphere at−20° C.

[0270] Slides carrying interphase cells and/or metaphase spreads areremoved from the nitrogen, heated to 65° C. for 4 hours in air, treatedwith RNase (100 micrograms/ml for 1 hour at 37° C.), and dehydrated inan ethanol series. They are then treated with proteinase K (60 ng/ml at37° C. for 7.5 minutes) and dehydrated. The proteinase K concentrationis adjusted depending on the cell type and enzyme lot so that almost nophase microscopic image of the chro-mosomes remains on the dry slide.The hybridization mix consists of (final concentrations) 50 percentformamide, 2× SSC, 10 percent dextran sulfate, 500 micrograms/ml carrierDNA (sonicated herring sperm DNA), and 2.0 microgram/ml biotin-labeledchromsome 21-specific DNA. This mixture is applied to the slides at adensity of 3 microliters/cm² under a glass coverslip and sealed withrubber cement. After overnight incubation at 37° C., the slides arewashed at 45° C. (50% formamide-2×SSC pH 7, 3 times 3 minutes; followedby 2×SSC pH 7, 5 times 2 minutes) and immersed in BN buffer (0.1 M Nabicarbonate, 0.05 percent NP-40, pH 8). The slides are never allowed todry after this point.

[0271] The slides are removed from the BN buffer and blocked for 5minutes at room temperature with BN buffer containing 5% non-fat drymilk (Carnation) and 0.02% Na Azide (5 microliter/cm² under plasticcoverslips). The coverslips are removed, and excess liquid brieflydrained and fluorescein-avidin DCS (3 microgram/ml in BN buffer with 5%milk and 0.02% NaAzide) is applied (5 microliter/cm²). The samecoverslips are replaced and the slides incubated. 20 minutes at 37° C.The slides are then washed 3 times for 2 minutes each in BN buffer at45° C. The intensity of biotin-linked fluorescence is amplified byadding a layer of biotinylated goat anti-avidin antibody (5 microgram/mlin BN buffer with 5% goat serum and 0.02% Na Azide), followed, afterwashing as above, by another layer of fluorescein-avidin DCS.Fluorescein-avidin DCS, goat antiavidin and goat serum are all availablecommercially, e.g., Vector Laboratories (Burlingame, CA). After washingin BN, a fluorescence antifade solution, p-phenylenediamine (1.5microliter/cm² of coverslip) is added before observation. It isimportant to keep this layer thin for optimum microscopic imaging. Thisantifade significantly reduced fluorescein fading and allows continuousmicroscopic observation for up to 5 minutes. The DNA counterstains (DAPIor propidium iodide) are included in the antifade at 0.25-0.5microgram/ml.

[0272] The red-fluorescing DNA-specific dye propidium iodide (PI) isused to allow simultaneous observation of hybridized probe and totalDNA. The fluorescein and PI are excited at 450-490 nm (Zeiss filtercombination 487709). Increasing the excitation wavelength to 546 nm(Zeiss filter combination 487715) allows observation of the PI only.DAPI, a blue fluorescent DNA-specific stain excited in the ultraviolet(Zeiss filter combination 487701), is used as the counterstain whenbiotin-labeled and total DNA are observed separately. Metaphasechromosome 21s are detected by randomly located spots of yellowdistributed over the body of the chromosome.

V.B. IMPROVED METHOD FOR EFFICIENTLY SELECTING CHROMOSOME 21 SINGLE-COPYSEQUENCES

[0273] Fuscoe et al., Genomics, 5:100-109 (1989) provides more efficientprocedures than the method described immediately above (V.A.2) forselecting large numbers of single-copy sequence or very low copy numberrepeat sequence clones from recombinant phage libraries and demonstratestheir use to stain chromosome 21. Said artide is hereby incorporated byreference. Briefly, clones were selected from the Charon 21A libraryLL21NS02 (made from DNA from human chromosome 21) using two basicprocedures. In the first, the phage library was screened in two stagesusing methods designed to be more sensitive to the presence ofrepetitive sequences in the clones than the method of Section V.A.2 .The selected clones were then subdoned into plasmids. The 450 insertsthus selected form the library pBS-U21. The second was in a multistepprocess in which: 1) Inserts from LL21NS02 were subdoned into Bluescribeplasmids, 2) plasmids were grown at high density in bacterial colonieson nitrocellulose filters and 3) radioactive human genomic DNA washybridized to the plasmid DNA on nitrocellulose filters at lowstringency in two steps and 4) plasmids having inserts that failed tohybridize were selected as potentially carrying single-copy sequences.Fifteen hundred and thirty colonies were picked in this manner to formthe library pBS-U21/1530.

[0274] Southern analysis indicated that the second procedure was moreeffective at recognizing repetitive sequence than the first.Fluorescence in situ hybridization with DNA from pBS-U21/1530 allowedspecific, intense staining of the number 21 chromosomes in metaphasespreads made from human lymphocytes. Hybridization with pBS-U21 givesless specific staining of chromosome 21. Details concerning the Fuscoeet al. method of selecting single-copy sequence or very low repeatsequence probes from recombinant libraries can be found in found inFuscoe et al., id.

V.C. HYBRIDIZATION WITH A COLLECTION OF CHROMOSOME 4 SINGLE-COPYSEQUENCES

[0275] Chromosome 4 Single-Copy Sequences. One hundred and twenty clonescarrying chromosome 4-specific single-copy sequence inserts selectedfrom the Charon 21A library LL04NS01 (ATCC accession number 57700; VanDilla et al., supra; see Table 1) were supplied by C. Gilliam (HarvardUniversity) [Gilliam et al., Nucleic Acids Res., 15:1445 (1987)]. Thehuman inserts were all about 3 kilobases (kb) in length, so the ratio ofinsert to vector DNA was <0.1. Total phage DNA was produced from eachclone individually using DEAE-cellulose columns (Whatman DE-52) [Helmset al., DNA, 4:39 (1985)]. DNA pooled from the 120 clones wasbiotinylated by nick-translation with biotin-11-dUTP (Bethesda ResearchLaboratories) and recovered at a concentration of about 20 nanograms permicroliter (ng/ul) using Sephadex G-50 spin columns.

[0276] Cells. Metaphase spreads from human lymphocytes were preparedfrom methotrexate-synchronized cultures by using the procedure of Harperet al., supra. The cells were fixed in methanol/acetic acid, 3:1. Slideswere stored at −20° C. in plastic bags filled with nitrogen gas.

[0277] In Situ Hybridization: Single-Copy Hybridization. Hybridizationwas accomplished by using a modification of the procedure described byPinkel et al., PNAS USA, 83: 2934 (1986). The slide mounted cells weretreated with RNase [100 micrograms per milliliter (ug/ml) in 0.3 molar(M) sodium chloride (NaCl)/30 millimolar (mM) sodium citrate at 37° C.for 1 hr), dehydrated in a 70%/85%/100% ethanol series, treated withproteinase K (0.3-0.6 ug/ml in 20 mM Tris/2 mM CaCl₂, pH 7.5, for 7.5min at 37° C.), and fixed 4% paraformaldehyde in phosphate-bufferedsaline (PBS; in g/liter, KCl, 0.2; KH₂PO₄, 0.2;NaCl,8;Na₂HPO₄.7H₂0,2.16)plus 50′ mM MgCl₂ for 10 min at room temperature]. The DNA in the targetcells was denatured by immersion in 70% formamide/2×SSC (0.3 M NaCI/30mM sodium citrate) at pH 7, for 2 min at 70° C. The hybridizationmixture [10 ul total volume consisting of 50% formamide, 0.3 M NaCl/30mM sodium citrate (final concentration), 10% dextran sulfate, 50 ug ofsonicated herring DNA per ml, and 3-6 ng of biotinylated chromosome 4unique sequences (40-80 ng of total phage DNA)] was then denatured (70°C. for 5 min) and applied. Hybridization was at 37° C. overnight (16hr). Slides were washed in three changes of 50% formamide/0.3 M NaCl/30mM sodium citrate (final concentration), pH 7, at 45° C. for 5 min eachand once in PN buffer (a mixture of 0.1 M NaH₂PO₄ and 0.1 M Na₂HPO₄ togive pH 8/0.1% Nonidet P-40). The slides were then treated withalternating layers of fluoresceinated avidin and biotinylated goatantiavidin, both at 5 ug/ml in PNM buffer (PN buffer/5% non-fat drymilk/0.02% sodium azide, centrifuged to remove solids), for 20 min eachat room temperature until three layers of avidin were applied. Theavidin and goat anti-avidin treatments were separated by three washes of3 min each in PN buffer [avidin (DCS grade) and anti-avidin from VectorLaboratories (Burlingame, Calif.)]. After the final avidin treatment, afluorescence antifade solution [Johnson and Noqueria, I. Immunol.Methods, 43:349 (1981)] containing 1 ug of 4′,6-amidino-2-phenylindoleor propidium iodide per ml was applied as a counterstain (1.5 ul/cm²under a no. 1 coverslip).

[0278] Results. As shown in FIG. 4H, individual hybridization sitescould be located to within a fraction of the width of a chromatid afterovernight hybridization (16 hr) and application of three layers ofavidin. Analysis of three spreads from the hybridization with the 120unique sequence probes at a total probe concentration of 1.5 pg/ul perkilobase of human insert, showed 222 fluorescent spots out of the 1440possible on the number 4 chromosomes (120 target sites per chromatid×4chromatids per metaphase×3 metaphases). Thus, the hybridizationefficiency was 15%. There were 814 total spots on all of the chromosomesgiving a hybridization specificity of 27%. The experiment demonstratesthat substantial hybridization can occur with single copy probes at lowprobe concentrations in overnight hybridizations. The contrast ratio ofchromosome 4 relative to the rest of the$\frac{\text{spots}/\text{length~~of~~chromosome~~4}}{\text{spots}/\text{length~~of~~all chromosomes}} = {\frac{222/{.06}}{814/1.0} = \text{approximately~~4.}}$

VI. INCAPACITATING SHARED REPETITIVE SEQUENCES VI.A. CHROMOSOME21-SPECIFIC STAINING USING BLOCKING DNA

[0279] High concentrations of unlabeled human genomic DNA and lambdaphage DNA were used to inhibit the binding of repetitive and vector DNAsequences to the target chromosomes. Heavy proteinase digestion andsubsequent fixation of the target improved access of probes to targetDNA.

[0280] Human metaphase spreads were prepared on microscope slides withstandard techniques and stored immediately in a nitrogen atmosphereat—20° C.

[0281] Slides were removed from the freezer and allowed to warm to roomtemperature in a nitrogen atmosphere before beginning the stainingprocedure. The warmed slides were first treated with 0.6 microgram/mlproteinase K in P buffer (20 mM Tris, 2 mM CaCl₂ at pH 7.5) for 7.5minutes, and washed once in P buffer. The amount of proteinase K usedneeds to be adjusted for different batches of slides. After denaturingthe slides were stored in 2×SSC. A hybridization mix was prepared whichconsisted of 50% formamide, 10% dextran sulfate, 1% Tween 20, 2×SSC, 0.5mg/ml human genomic DNA, 0.03 mg/ml lambda DNA, and 3 microgram/mlbiotin labeled probe DNA. The probe DNA consisted of the highest densityfraction of phage from the chromosome 21 Hind III fragment library (ATCCaccession number 57713), as determined by a cesium chloride gradient.(Both insert and phage DNA of the probe were labeled by nicktranslation.) The average insert size (amount of chromosome 21 DNA), asdetermined by gel electrophoresis was about 5 kilobases. No attempt wasmade to remove repetitive sequences from the inserts or to isolate theinserts from the lambda phage vector. The hybridization mix wasdenatured by heating to 70° C. for 5 minutes followed by incubation at37° C. for 1 hour. The incubation allows the human genomic DNA andunlabeled lambda DNA in the hybridization mix to block the humanrepetitive sequences and vector sequences in the probe.

[0282] The slide containing the human metaphase spread was removed fromthe 2×SSC and blotted dry with lens paper. The hybridization mix wasimmediately applied to the slide, a glass cover slip was placed on theslide with rubber cement, and the slide was incubated overnight at 37°C. Afterwards preparation of the slides proceeded as described inSection V.B. (wherein chromosome 21 DNA was stained with fluorescein andtotal chromosomal DNA counterstained with DAPI). FIGS. 1A-C illustratethe results. FIG. 1A is a DAPI image of the human metaphase spreadobtained with a computerized image analysis system. It is a binary imageshowing everything above threshold as white, and the rest as black. Theprimary data was recorded as a gray level image with 256 intensitylevels. (Small arrows indicate the locations of the chromosome 21s.)FIG. 1B is a fluorescein image of the same spread as in FIG. 1A, againin binary form. (Again, small arrows indicate the locations of thechromosome 21s.) FIG. 1C illustrates the positions of the chromosome 21safter other less densely stained objects were removed by standard imageprocessing techniques.

VI.B. DETECTION OF TRISOMY 21 AND TRANSLOCATIONS OF CHROMOSOME 4 USINGBLUESCRIBE PLASMID LIBRARIES

[0283] As illustrated in Section VI.A., a human chromosome-specificlibrary, including its shared repetitive sequences, can be used to stainthat chromosome if the hybridization capacity of the shared repetitivesequences is reduced by incubation with unlabeled human genomic DNA. InSection VI.A., the nucleic acid sequences of the heterogeneous mixturewere cloned in the phage vector Charon 21A, in which the ratio of insertof vector DNA is about 0.1 (4 kb average insert to 40 kb of vector). Inthis section, we demonstrate that transferring the same inserts to asmaller cloning vector, the about 3 kb Bluescribe plasmid, whichincreases the ratio of insert to vector DNA to 0.5, improved thespecificity and intensity of the staining.

[0284] As previously discussed, incubation of the probe can be carriedout with the probe alone, with the probe mixed with unlabeled genomicDNA, and with the probe mixed with unlabeled DNA enriched in all or someshared repetitive sequences. If unlabeled genomic DNA is added, then itis important to add enough to incapacitate sufficiently the sharedrepetitive sequences in the probe. However, the genomic DNA alsocontains unlabeled copies of the sequences, the hybridization of whichis desired. As explained above, Q is herein defined as the ratio ofunlabeled to labeled copies of the chromosome-specific sequences in thehybridization mixture.

[0285] Cells. Metaphase spreads from human lymphocytes were preparedfrom methotrexate-synchronized cultures by using the procedure of Harperet al. supra. These and all other cells used in this example were fixedin methanol/acetic acid, 3:1. Other human lymphocyte cultures wereirradiated with ⁶⁰Co gamma rays and stimulated with phytohemagglutnin.Colcemid was added 48 hr after stimulation and metaphase spreads wereprepared 4 hr later. Metaphase spreads and interphase cells fromlymphoblastoid cells (GM03716A; Human Mutant Cell Repository, Camden,N.J.) carrying trisomy 21 were prepared after a 4-hr colcemid block.Interphase cells from the cell line RS4;11 carrying t(4;11) andisochromosome 7q were harvested, fixed in methanol/acetic add, anddropped onto slides [Strong et al., Blood, 65:21 (1985)]. Slides werestored at −20° C. in plastic bags filled with nitrogen gas.

[0286] pBS-4. The entire chromosome 4 library LL04NS02 (ATCC accessionnumber 57745; Van Dilla et al., supra) was subcloned into Bluescribeplasmids (Stratagene La Jolla, Calif.) to form the library pBS-4. Theaverage insert to vector DNA ratio in pBS-4 is about 1. The plasmidlibrary was amplified in bulk and the DNA was extracted usingDEAE-cellulose columns (Whatman DE-52) [Helms et al., DNA, 4:39 (1985)].The DNA was then biotinylated by nick translation with biotin-11-dUTP(Bethesda Research Laboratories) and recovered at a concentration ofabout 20 ng/ul using Sephadex G-50 spin columns. In some experiments,the biotinylated DNA was concentrated by ethanol precipitation toachieve higher probe concentrations.

[0287] pBS-21. The entire chromosome 21 library LL21NS02 (ATCC accessionnumber 57713; Van Dilla et al., supra) was subdoned into Bluescribeplasmids to form the library pBS-21. This library was amplified andbiotinylated as described above for pBS-4.

[0288] Human genomic DNA. Placental DNA (Sigma) was treated withproteinase K, extracted with phenol, and sonicated to a size range of200-600 base pairs (bp).

[0289] Whole Library Hybridization. Hybridization was as above insection V.C except that RNase, proteinase K, and paraformaldehyde werenot used. The amount of probe and genomic DNA in the hybridizationmixture and the length of the hybridization varied as described inResults. All probe concentrations refer to the human insert DNA unlessotherwise noted. DNA concentrations were determined by fluorometricanalysis (Hoeffer Scientific Instruments, San Francisco). Incubation ofthe hybridization mixture prior to hybridization followed two differentprotocols as indicated immediately below.

[0290] Protocol I. The hybridization mixture (10 ul) contained 10-150 ngof biotinylated human DNA (20-300 ng of total plasmid DNA) and 0-10 ugof unlabeled genomic DNA. The mixture was heated to denature the DNA andincubated at 37° C. for a time t before it was added to the slide.Hybridization times ranged from 2 to 110 hr.

[0291] Protocol II. Protocol II was identical to Protocol I except thatan additional aliquot of freshly denatured genomic DNA was added to thehybridization mixture after an incubation time t. The mixture was thenincubated an additional time t prior to starting the hybridization. Thevolume of the hybridization mixture was increased <20% by the additionalgenomic DNA.

[0292] Microscopy. Quantitative fluorescence measurements were performedusing a video camera on the microscope and a digital image processingsystem, [Trask et al., Human Genet., 78:251 (1988)]

[0293] Results. FIG. 4A shows hybridization of pBS-4 to a humanmetaphase spread with a probe concentration of 1 ng/ul. No genomic DNAwas used and the hybridization mixture was applied immediately afterdenaturation. All of the chromosomes are stained, except near manycentromeres, with two copies of chromosome 4 being stained most heavily.All the chromosomes are stained along most of their lengths due tosequences in the probe which are shared with other chromosomes.Unstained regions, noted by arrows, show locations for which homologoussequences are not present in pBS-4. The unstained regions are mostlycentromeric and along the long arm of the Y chromosome. Blocks ofrepetitive DNA specific to those sites are known to exist.

[0294] The visible contrast on chromosome 4 is the result of theinteraction of several factors. (i) All of the DNA in chromosome 4 ispotential target for sequences in the probe, whereas only thosesequences on the other chromosomes that are shared with chromosome 4 canbind probe. (ii) The hybridization time and probe concentration werehigh enough to allow significant binding of the specific sequences inthe probe. (iii) The ratio of probe to target sequences is higher forthe specific sequences than for the shared sequences [Ten nanograms ofchromosome 4 DNA was hybridized to about 200 ng of human DNA target(4×10⁴ cells), 13 ng of which is chromosome 4. Thus, the ratio of probeto target for the specific sequences was about 1, whereas for the sharedsequences it was about 0.05.]

[0295] The contrast can be increased by allowing the denatured probe DNAto partially reassociate prior to adding it to the slide, preferentiallydepleting the single-stranded high-copy (predominantly the shared)sequences in the probe [Cantor & Schimmel, Biophysical Chemistry: TheBehavior of Biological Macromolecules, (part III, p. 1228) (Freeman1980)]. A significant increase in staining specificity resulting fromprobe reassociation was observed experimentally for chromosome 4 using ahybridization mixture with 1 ng of probe per microliter (ul) and a 24-hrincubation at 37° C. prior to in situ hybridization (not shown).Likewise, hybridization after a 24 hr incubation of 4 ng of chromosome21 probe per ul resulted in a substantial contrast ratio. That resultindicates that at such concentrations the chromosome-specific sequencesremain substantially single stranded for times on the order of days inthe hybridization mixture. It also demonstrates that other mechanismsthat might inactivate the probe are not significant during theincubation.

[0296]FIGS. 4B and 4C show the result of a protocol I hybridization [0.8ng of probe per ul and 24 ng of genomic DNA per ul (Q=2); 1-hr probeincubation and 110-hr hybridization]. Quantitative image analysis showsthat the intensity per unit length of the FITC fluorescein on chromosome4 is approximately 20 times that of the other chromosomes, that is thecontrast ratio is 20:1. Two layers of avidin-fluorescein isothiocyanatehave been used here to make the non-target chromosomes sufficientlybright to be measured accurately. However, the number 4 chromosomes canbe recognized easily after a single layer.

[0297]FIG. 4D demonstrates detection of a radiation-inducedtranslocation involving chromosome 4 in human lymphocytes [protocol I,1′ ng of probe per ul and 76 ng of genomic DNA per ul (Q=5); 1-hr probeincubation and 16-hr hybridization]. The contrast ratio was about 5. Thehybridization intensity and specificity shown in FIG. 4D are such thateven small portions of the involved chromosome can be detected.

[0298] The ease with which translocations can be recognized offers theopportunity for translocation detection by automated means, such as, 2 0computerized microscopy or flow cytometry. [See Section vm infra forelaboration concerning automated detection means.]

[0299]FIG. 4E shows that the normal and two derivative chromosomesresulting from the translocation between chromosomes 4 and 11 [t(4;11)]in cell line RS4;11 can be detected in interphase nuclei as threedistinct domains [protocol I, 13.5 ng of probe per ul and 800 ng ofgenomic DNA per ul (Q=5); 1-hr probe incubation and 16-hrhybridization]. The increased probe concentration resulted in brightersignals relative to FIG. 4D. Approximately half of the cells clearlyshow the presence of three nuclear domains, presumably produced by thetwo portions of the involved chromosome 4 and the intact normalchromosome. The domains in the other nuclei may have been obscured bythe nuclear orientation in these two-dimensional views, by nucleardistortion that occurred during slide preparation, or because thedomains were too close to each other to be distinguished. Hybridizationusing procedures that preserve three-dimensional morphology may resolvethese issues and also permit general studies of chromosomal domains ininterphase nuclei [Trask et al., Hum. Genet., 78:251 (1988)].

[0300] Hybridization of pBS-21 to a metaphase spread from a cell linewith trisomy 21 is shown in FIG. 4F [protocol II, 4 ng of probe per uland 250 ng of genomic DNA per ul; 3-hr incubation, additional 250 ng ofgenomic DNA per ul (Q=1+1); 3-hr probe incubation and 16-hrhybridization]. A small amount of hybridization is visible near thecentromeres of the other acrocentric chromosomes.

[0301]FIG. 4G shows two interphase nuclei from the same hybridizationwhich clearly show the three chromosome 21 domains. Hybridization withprobe prepared according to protocol I resulted in higher relativeintensity of the shared signals on the D- and G-group chromosomes, andconsequently it was more difficult to determine the number of number 21chromosomes in interphase (not shown). Increasing stringency by using ahybridization mixture with 55% formamide and 0.15 M NaCl/15 mM sodiumcitrate, which lowers the melting temperature about 8° C., did notreduce the unwanted hybridization. Addition of unlabeled pA ribosomalDNA [Erikson et al., Gene, 16:1 (1981)] also was ineffective atincreasing specificity.

[0302] The centromeric region of the D- and G-group chromosomes containribosomal [Erikson et al., id] and alpha satellite sequences and perhapsothers [Choo et al., Nucleic Acids Res., 16: 1273 (1988)]. These arerelatively low copy sequences shared with only a few chromosomes, soProtocol I is not very effective at suppressing them relative to thechromosome 21-specific sequences. In addition, these sequences areclustered on the chromosomes, so that even much reduced hybridization isclearly visible. This is especially distracting in analysis ofinterphase nuclei. Calculations indicate that addition of severalaliquots of freshly denatured genomic DNA periodically during theincubation (protocol II) should increase the staining specificity. FIG.4F shows a protocol II hybridization, using two aliquots of genomic DNA,to a metaphase spread from a trisomy 21 cell line. Intense hybridizationto the three number 21 chromosomes is clearly visible and hybridizationto the other D- and G-group chromosomes has been reduced to anacceptable level. FIG. 4G shows that hybridization to chromosomes otherthan chromosome 21 is sufficiently low that the three chromosome 21domains are clearly visible in interphase nuclei. In practice, the mostconvenient procedure for suppressing the shared acrocentrichybridization might be inclusion of unlabeled DNA from one of the otherD- or G-group chromosome libraries (or unlabeled cloned DNA from justthese sequences, if available) as additional competitor. The use oflibraries from non-target chromosomes as blocker for a probe may ingeneral improve contrast. The specific sequences in the probe will notbe blocked (Q=0) no matter how much competitor for the shared sequencesis added.

VI.C. HYBRIDIZATION OF YEAST ARTIFICIAL CHROMOSOMES (YACS) TO HUMANMETAPHASE SPREAD

[0303] YACS. Seven yeast clones HY1, HY19, HY29, HYA1.A2, HYA3.A2,HYA3.A9, and HYA9.E6 were obtained from D. Burke (Washington University,St. Louis, Mo.). The lengths of the human DNA in the clones ranged fromabout 100 kb to about 600 kb. Gel electrophoresis was performed toverify the size of these inserts. Each of these clones was grown up andtotal DNA was isolated. The isolated DNA was biotinylated by nicktranslation so that 10-30% of the thymidine was replaced bybiotin-11-dUTP. The concentration of the total labeled DNA after nicktranslations is in the range of 10-20 ng/ul.

[0304] Blocking DNA. Human placental DNA (Sigma) was treated withproteinase K and extracted with phenol and sonicated to a size range of200-600 bp. Total DNA isolated from yeast not containing an artificialchromosome was sonicated to a similar size range. Both of these DNA'swere maintained at a concentration of 1-10 ug/ul.

[0305] Fluorescence in situ hybridization (FISH). Hybridization followedthe procedures of Pinkel et al. (1988), supra (as exemplified inSections V and VI, supra) with slight modifications. Metaphase spreadswere prepared from methotrexate synchronized cultures according to theprocedures of Harper et al. PNAS (USA) 78: 4458-4460, (1981). Cells werefixed in methanol/acetic acid, fixed (3:1), dropped onto slides, airdried, and stored at −20° C. under nitrogen gas until used. The slideswere then immersed two minutes in 70% formamide/2×SSC to denature thetarget DNA sequences, dehydrated in a 70-85-100% ethanol series, and airdried. (SSC is 0.15 M NaCl/0.015 M'Na Citrate, pH 7). Ten-100 ng ofbiotinylated yeast DNA, and approximately 1 ug each of unlabeled yeastand human genomic DNA were then added to the hybridization mix (finalvolume 10 ul, final composition 50% formamide/2×SSC/10% dextransulfate), heated to 70° C. for 5 min., and then incubated at 37° C. for1 hr to allow the complementary strands of the more highly repeatedsequences to reassociate.

[0306] The hybridization mixture was then applied to the slide(approximately 4 cm² area) and sealed with rubber cement under a glasscover slip. After overnight incubation at 37° C. the coverslip wasremoved and the slide washed 3 times 3 min each in 50% formamide/2×SSCat 42-45° C., and once in PN buffer [mixture of 0.1 M NaH₂PO₄ and 0.1 MNa₂HPO₄ to give pH 8; 0.1% Nonidet P40 (Sigma)]. The bound probe wasthen detected with alternating 20 min incubations (room temperature inavidin-F ITC and goat-anti-avidin antibody, both at 5 ug/ml in PNMbuffer (PN buffer plus 5% nonfat dry milk, centrifuged to remove solids;0.02% Na azide). Avidin and anti-avidin incubation were separated by 3washes of 3 min each in PN buffer. Two or three layers of avidin wereapplied (Avidin, DCS grade, and biotinylated goat-anti-avidin areobtained from Vector Laboratories Inc., Burlingame, Calif.).

[0307]FIG. 5 shows the hybridization of HYA3.A2 (580 kb of human DNA) to12q21.1. The location of the hybridization was estabilished by using aconventional fluorescent banding technique employing theDAPI/actinomycin D procedure: Schweizer, “Reverse fluorescent chromosomebanding with chromomycin and DAPI,” Chromosoma 58:307-324 (1976). Thehybridization signal forms a band across the width of each of thechromosome 12s, indicating the morphology of the packing of DNA in thatregion of the chromosome.

[0308] The YAC clone positions are attributed as shown in Table 2 below.TABLE 2 YAC Competition Hybridization YAC Clone Insert Size LocalizationHY1 120 Xq23 HY19 450 8q23.3 21q21.1 HY29 500 14q12 HYA1.A2 250 6q16HYA3.A2 580 12q21.1 HYA3.A9 600 14q21 HYA9.E6 280 1p36.2 3q22

VI.D. HYBRIDIZATION WITH HUMAN/HAMSTER HYBRID CELL

[0309] Essentially the same hybridization and staining conditions wereused in this example as for those detailed in the procedure of Pinkel etal. (1988), supra and exemplified in Sections V.C. and VI.B., supra. Inthis example, 400 ng of biotin labeled DNA from a hamster-human hybridcell that contains one copy of human chromosome 19 was mixed with 1.9 ugof unlabeled human genomic DNA in 10 ul of hybridization mix.Hybridization was for approximately 60 hours at 37° C. Fluorescentstaining of the bound probe and counterstaining of the chromosomes wasas in the other examples above. FIG. 6 shows the results of thehybridization.

VII. SPECIFIC APPLICATIONS

[0310] The present invention allows microscopic and in some cases flowcytometric detection of genetic abnormalities on a cell by cell basis.The microscopy can be performed entirely by human observers, or includevarious degrees of addititional instrumentation and computationalassistance, up to full automation. The use of instrumentation andautomation for such analyses offers many advantages. Among them are theuse of fluorescent dyes that are invisible to human observers (forexample, infared dyes), and the opportunity to interpret resultsobtained with multiple labeling methods which might not besimultaneously visible (for example, combinations of fluorescent andabsorbing stains, autoradiography, etc.) Quantitative measurements canbe used to detect differences in staining that are not detectable byhuman observers. As is described below, automated analysis can alsoincrease the speed with which cells and chromosomes can be analysed.

[0311] The types of cytogenetic abnormalities that can be detected withthe probes of this invention include: Duplication of all or part of achromosome type can be detected as an increase in the number or size ofdistinct hybridization domains in metaphase spreads or interphase nucleifollowing hybridization with a probe for that chromosome type or region,or by an increase in the amount of bound probe. If the probe is detectedby fluorescence, the amount of bound probe can be determined either flowcytometrically or by quantitative fluorescence microscopy. Deletion of awhole chromosome or chromosome region can be detected as a decrease inthe number or size of distinct hybridization domains in metaphasespreads or interphase nuclei following hybridization with a probe forthat chromosome type or region, or by a decrease in the amount of boundprobe. If the probe is detected by fluorescence, the amount bound can bedetermined either flow cytometrically or by quantitative fluorescencemicroscopy. Translocations, dicentrics and inversions can be detected inmetaphase spreads and interphase nuclei by the abnormal juxtaposition ofhybridization domains that are normally separate following hybridizationwith probes that flank or span the region(s) of the chromosome(s) thatare at the point(s) of rearrangement. Translocations involve at leasttwo different chromosome types and result in derivative chromosomespossessing only one centromere each. Dicentrics involve at least twodifferent chromosome types and result in at least one chromosomefragment lacking a centromere and one having two centromeres. Inversionsinvolve a reversal of polarity of a portion of a chromosome.

VII.A BANDING ANALYSIS

[0312] Substantial effort has been devoted during the past thirty yearsto development of automated systems (especially computer controlledmicroscopes) for automatic chromosome classification and aberrationdetection by analysis of metaphase spreads. In recent years, effort hasbeen directed at automatic classification of chromosomes which have beenchemically stained to produce distinct banding patterns on the variouschromosome types. These efforts have only partly succeeded because ofthe subtle differences in banding pattern between chromosome types ofapproximately the same size, and because differential contraction ofchromosomes in different metaphase spreads causes a change in the numberand width of the bands visible on chromosomes of each type. The presentinvention overcomes these problems by allowing construction of reagentswhich produce a staining pattern whose spacing, widths and labelingdifferences (for example different colors) are optimized to facilitateautomated chromosome classification and aberration detection. This ispossible because hybridization probes can be selected as desired alongthe lengths of the chromosomes. The size of a band produced by such areagent may range from a single small dot to a substantially uniformcoverage of one or more whole chromosomes. Thus the present inventionallows construction of a hybridization probe and use of labeling means,preferably fluorescence, such that adjacent hybridization domains can bedistinguished, for example by color, so that bands too closely spaced tobe resolved spatially can be detected spectrally (i.e. if red and greenfluorescing bands coalesce, the presence of the two bands can bedetected by the resulting yellow fluorescence).

[0313] The present invention also allows construction of bandingpatterns tailored to particular applications. Thus they can besignificantly different in spacing and color mixture, for example, onchromosomes that are similar in general shape and size and which havesimilar banding patterns when conventional techniques are used. Thesize, shape and labeling (e.g. color) of the hybridization bandsproduced by the probes of the present invention can be optimized toeliminate errors in machine scoring so that accurate automatedaberration detection becomes possible. This optimized banding patternwill also greatly improve visual chromosome classification andaberration detection.

[0314] The ease of recognition of specific translocation breakpoints canbe improved by using a reagent closely targeted to the region of thebreak. For example, a high complexity probe of this invention comprisingsequences that hybridize to both sides of the break on a chromosome canbe used. The portion of the probe that binds to one side of the breakcan be detected differently than that which binds to the other, forexample with different colors. In such a pattern, a normal chromosomewould have the different colored hybridization regions next to eachother, and such bands would appear close together. A break wouldseparate the probes to different chromosomes or result in chromosomalfragments, and could be visualized as much further apart on an average.

VII.B BIOLOGICAL DOSIMETRY

[0315] One approach to biological dosimetry is to measure frequencies ofstructurally aberrant chromosomes as an indication of the genetic damagesuffered by individuals exposed to potentially toxic agents. Numerousstudies have indicated the increase in structural aberration frequencieswith increasing exposure to ionizing radiation and other agents, whichare called clastogens. Dicentric chromosomes are most commonly scoredbecause their distinctive nature allows them to be scored rapidlywithout banding analysis. Rapid analysis is important because of the lowfrequency of such aberrations in individuals exposed at levels found inworkplaces (˜2×10⁻³/cell). Unfortunately, dicentrics are not stablyretained so the measured dicentric frequency decreases with time afterexposure. Thus low level exposure over long periods of time does notresult in an elevated dicentric frequency because of the continuedclearance of these aberrations. Translocations are better aberrations toscore for such dosimetric studies because they are retained more or lessindefinitely. Thus, assessment of genetic damage can be made at timeslong after exposure. Translocations are not routinely scored forbiological dosimetry because the difficulty of recognizing them makesscoring sufficient cells for dosimetry logistically impossible.

[0316] The present invention eliminates this difficulty. Specifically,hybridization with a probe which substantially uniformly stains severalchromosomes (e.g. chromosomes 1, 2, 3 and 4) allows immediatemicroscopic identification in metaphase spreads of structuralaberrations involving these chromosomes. Normal chromosomes appearcompletely stained or unstained by the probe. Derivative chromosomesresulting from translocations between targeted and non-targetedchromosomes are recognized as being only partly stained, FIG. 4D. Suchpartially hybridized chromosomes can be immediately recognized eithervisually in the microscope or in an automated manner using computerassisted microscopy. Discrimination between translocations anddicentrics is facilitated by adding to the probe, sequences found at allof the chromosome centromeres. Detection of the centromeric componentsof the probe with a labeling means, for example color, different fromthat used to detect the rest of the probe elements allows readyidentification of the chromosome centromeres, which in turn facilitatesdiscrimination between dicentrics and translocations. This technologydramatically reduces the scoring effort required with previoustechniques so that it becomes feasible to examine tens of thousands ofmetaphase spreads as required for low level biological dosimetry.

VII.C. PRENATAL DIAGNOSIS

[0317] The most common aberrations found prenatally are trisomiesinvolving chromosomes 21 (Down syndrome), 18 (Edward syndrome) and 13(Patau syndrome) and X0 (Turner syndrome), XXY (Kleinfelter syndrome)and XYY disease. Structural aberrations also occur. However, they arerare and their clinical significance is often uncertain. Thus, theimportance of detecting these aberrations is questionable. Currenttechniques for obtaining fetal cells for conventional karyotyping, suchas, amniocentesis and chorionic villus biopsy yield hundreds tothousands of cells for analysis. These are usually grown in culture for2 to 5 weeks to produce sufficient mitotic cells for cytogeneticanalysis. Once metaphase spreads are prepared, they are analyzed byconventional banding analysis. Such a process can only be carried out byhighly skilled analysts and is time consuming so that the number ofanalyses that can be reliably carried out by even the largestcytogenetics laboratories is only a few thousand per year. As a result,prenatal cytogenetic analysis is usually limited to women whose childrenare at high risk for genetic disease (e.g. to women over the age of 35).

[0318] The present invention overcomes these difficulties by allowingsimple, rapid identification of common numerical chromosome aberrationsin interphase cells with no or minimal cell culture. Specifically,abnormal numbers of chromosomes 21, 18, 13, X and Y can be detected ininterphase nuclei by counting numbers of hybridization domains followinghybridization with probes specific for these chromosomes (or forimportant regions thereof such as 21q22 for Down syndrome). Ahybridization domain is a compact, distinct region over which theintensity of hybridization is high. An increased frequency of cellsshowing three domains (specifically to greater than 10%) for chromosomes21, 18 and 13 indicates the occurrence of Down, Edward and Patausyndromes, respectively. An increase in the number of cells showing asingle X-specific domain and no Y-specific domain followinghybridization with X-specific and Y-specific probes indicates theoccurrence of Turner syndrome. An increase in the frequency showing twoX-specific domains and one Y-specific domain indicates Kleinfeltersyndrome, and increase in the frequency of cells showing one X-specificdomain and two Y-specific domains indicates an XYY fetus. Domaincounting in interphase nuclei can be supplemented (or in some casesreplaced) by measurement of the intensity of hybridization using, forexample, quantitative fluorescence microscopy or flow cytometry, sincethe intensity of hybridization is approximately proportional to thenumber of target chromosomes for which the probe is specific. Numericalaberrations involving several chromosomes can be scored simultaneouslyby detecting the hybridization of the different chromosomes withdifferent labeling means, for example, different colors. Theseaberration detection procedures overcome the need for extensive cellculture required by procedures since all cells in the population can bescored. They eliminate the need for highly skilled analysts because ofthe simple, distinct nature of the hybridization signatures of numericalaberrations. Further, they are well suited to automated aberrationanalysis.

[0319] The fact that numerical aberrations can be detected in interphasenuclei also allows cytogenetic analysis of cells that normally cannot bestimulated into mitosis. Specifically, they allow analysis of fetalcells found in maternal peripheral blood. Such a feature is advantageousbecause it eliminates the need for invasive fetal cell sampling such asamniocentesis or chorionic villus biopsy.

[0320] As indicated in the Background, the reason such embryo-invasivemethods are necessary is that conventional karyotyping and bandinganalysis requires metaphase chromosomes. At this time, there are noaccepted procedures for culturing fetal cells separated from maternalblood to provide a population of cells having metaphase chromosomes. Inthat the staining reagents of this invention can be employed withinterphase nuclei, a non-embryo-invasive method of karyotyping fetalchromosomes is provided by this invention.

[0321] The first step in such a method is to separate fetal cells thathave passed through the placenta or that have been shed by the placentainto the maternal blood. The incidence of fetal cells in the maternalbloodstream is very low, on the order of 10⁻⁴ to 10⁻⁶ cells/ml and quitevariable depending on the time of gestation; however, appropriatelymarked fetal cells may be distinguished from maternal cells andconcentrated, for example, with high speed cell sorting.

[0322] The presence of cells of a male fetus may be identified by alabel, for example a fluorescent tag, on a chromosome-specific stainingreagent for the Y chromosome. Cells that were apparently eitherlymphocytes or erythrocyte precursors that were separated from maternalblood were shown to be Y-chromatin-positive. [Zillacus et al., Scan. J.Haematol, 15: 333 (1975); Parks and Herzenberg, Methods in Cell Biology,Vol. 10, pp. 277-295 (Academic Press, N.Y., 1982); and Siebers et al.,Humangenetik, 28: 273 (1975)].

[0323] A preferred method of separating fetal cells from maternal bloodis the use of monoclonal antibodies which preferentially have affinityfor some component not present upon the maternal blood cells. Fetalcells may be detected by paternal HLA (human leukocyte antigen) markersor by an antigen on the surface of fetal cells. Preferred immunochemicalprocedures to distinguish-between fetal and maternal leukocytes on thebasis of differing HLA type use differences at the HLA-A2, -A3, and -B7loci, and further preferred at the -A2 locus. Further, first and secondtrimester fetal trophoblasts may be marked with antibody against theinternal cellular constituent cytokeratin which is not present inmaternal leukocytes. Exemplary monoclonal antibodies are described inthe following references.

[0324] Herzenberg et al., PNAS, 76: 1453 (1979), reports the isolationof fetal cells, apparently of lymphoid origin, from maternal blood byfluorescence activated cell sorting (FACS) wherein the separation wasbased on the detection of labeled antibody probes which bind HLA-A2negative cells in maternal blood. Male fetal cells separated in thatmanner were further identified by quinacrine staining of Y-chromatin.

[0325] Covone et al., Lancet, Oct. 13, 1984: 841, reported the recoveryof fetal trophoblasts from maternal blood by flow cytometry using amonoclonal antibody termed H315. Said monoclonal reportedly identifies aglycoprotein expressed on the surface of the human syncytiotrophoblastas well as other trophoblast cell populations, and that is absent fromperipheral blood cells.

[0326] Kawata et al., J. Exp. Med., 160:653 (1984), discloses a methodfor isolating placental cell populations from suspensions of humanplacenta. The method uses coordinate two-color and light-scatter FACSanalysis and sorting. Five different cell populations were isolated onthe basis of size-and quantitative differences in the coordinateexpression of cell surface antigens detected by monoclonal antibodiesagainst an HLA-A, B, C monomorphic determinant (MB40.5) and againsthuman trophoblasts (anti-Trop-1 and anti-Trop-2).

[0327] Loke and Butterworth, J. Cell Sci., 76: 189 (1985), describe twomonoclonal antibodies, 18B/A5 and 18A/C4, which are reactive with firsttrimester cytotrophoblasts and other fetal epithelial tissues includingsyncytiotrophoblasts.

[0328] A preferred monoclonal antibody to separate fetal cells frommaternal blood for staining according to this invention is theanti-cytokeratin antibody Cam 5.2, which is commercially available fromBecton-Dickinson (Franklin Lakes, N.J., USA).

[0329] Other preferred monoclonal antibodies for separating fetal cellsfrom maternal blood are those disclosed in co-pending, commonly ownedU.S. patent application Ser. No. 389,224, filed Aug. 3, 1989, entitled“Method for Isolating Fetal Cytotrophoblast Cells”. [See also: in Fisheret al., J. Cell. Biol., 109 (2): 891-902 (1989)]. The monoclonalantibodies disclosed therein react specifically with antigen on firsttrimester human cytotrophoblast cells, which fetal cells have thehighest probability of reaching the maternal circulation. Saidapplication and article are herein specifically incorporated byreference. Briefly, the disclosed monoclonal antibodies were raised byinjection of test animals with cytotrophoblast cells obtained fromsections of the placental bed, that had been isolated by uterineaspiration. Antibodies raised were subjected to several cytologicalscreens to select for those antibodies which react with thecytotrophoblast stem cell layer of first trimester chorionic villi.

[0330] Preferred monoclonal antibodies against such first trimestercytotrophoblast cells disclosed by Fisher et al. include monoclonalantibodies produced from the following hybridomas deposited at theAmerican Tyupe Culture Collection (ATCC; Rockville, Md., USA) under theBudapest Treaty: Hybridoma ATCC Accession # J1D8 HB10096 P1B5 HB10097

[0331] Both hybridoma cultures were received by the ATCC on Apr. 4, 1989and reported viable thereby on Apr. 14, 1989.

[0332] Fisher et al. state that fetal cells isolated from maternal bloodby use of said monoclonal antibodies are capable of replication invitro. Therefore, fetal cells isolated by the method of Fisher et al.,that is, first trimester fetal cytotrophoblasts, may provide fetalchromosomal material that is both in metaphase and in interphase.

[0333] The fetal cells, preferably leukocytes and cytotrophoblasts, morepreferably cytotrophoblasts, once marked with an appropriate antibodyare then separated from the maternal cells either directly or bypreferably separating and concentrating said fetal cells by cell sortingor panning. For example, FACS may be used to separate fluorescentlylabeled fetal cells, or flow cytometry may be used.

[0334] The fetal cells once separated from the maternal blood can thenbe stained according to the methods of this invention with appropriatechromosome-specific staining reagents of this invention, preferablythose of particular importance for prenatal diagnosis. Preferredstaining reagents are those designed to detect aneuploidy, for example,trisomy of any of several chromosomes, including chromosome types 21,18, 13, X and Y and subregions on such chromosomes, such as, subregion21q22 on chromosome 21.

[0335] Preferably, a fetal sample for staining analysis according tothis invention comprises at least 10 cells or nuclei, and morepreferably about 100 cells or nuclei.

VII.D Tumor Cytogenetics

[0336] Numerous studies in recent years have revealed the existence ofstructural and numerical chromosome aberrations that are diagnostic forparticular disease phenotypes and that provide clues to the geneticnature of the disease itself. Prominent examples include the closeassociation between chronic myelogeneous leukemia and a translocationinvolving chromosome 9 and 22, the association of a deletion of aportion of 13q14 with retinoblastoma and the association of atranslocation involving chromosomes 8 and 14 with Burkitts lymphoma.Current progress in elucidating new tumor specific abnormalities islimited by the difficulty of producing representative, high qualitybanded metaphase spreads for cytogenetic analysis. These problems stemfrom the fact that many human tumors are difficult or impossible to growin culture. Thus, obtaining mitotic cells is usually difficult. Even ifthe cells can be grown in culture, there is the significant risk thatthe cells that do grow may not be representative of the tumorigenicpopulation. That difficulty also impedes the application of existinggenetic knowledge to clinical diagnosis and prognosis.

[0337] The present invention overcomes these limitations by allowingdetection of specific structural and numerical aberrations in interphasenuclei. These aberrations are detected as described supra. Hybridizationwith whole chromosome probes will facilitate identification ofpreviously unknown aberrations thereby allowing rapid development of newassociations between aberrations and disease phenotypes. As the geneticnature of specific malignancies becomes increasingly well known, theinterphase assays can be made increasingly specific by selectinghybridization probes targeted to the genetic lesion. Translocations atspecific sites on selected chromosomes can be detected by usinghybridization probes. that closely flank the breakpoints. Use of theseprobes allows diagnosis of these specific disease phenotypes.Translocations may be detected in interphase because they bring togetherhybridization domains that are normally separated, or because theyseparate a hybridization domain into two, well separated domains. Inaddition, they may be used to follow the reduction and reemergence ofthe malignant cells during the course of therapy. Interphase analysis isparticularly important in such a application because of the small numberof cells that may be present and because they may be difficult orimpossible to stimulate into mitosis.

[0338] Duplications and deletions, processes involved in geneamplification and loss of heterozygosity, can also be detected inmetaphase spreads and interphase nuclei using the techniques of thisinvention. Such processes are implicated in an increasing number ofdifferent tumors.

VIII. DETECTION OF BCR-ABL FUSION IN CHRONIC MYELOGENOUS LEUKEMIA (CML)

[0339] Probes. This section details a CML assay based upon FISH withprobes from chromosomes 9 and 22 that flank the fused BCR and ABLsequences in essentially all cases of CML (FIG. 8). The BCR and ABLprobes used in the examples of this section were kindly provided byCarol A. Westbrook of the Department of Medicine, Section ofHematology/Oncology at the University of Chicago Medical Center inChicago, Ill. (USA).

[0340] The ABL probe on chromosome 9, c-hu-ABL, is a 35-kb cosmid(pCV105) clone selected to be telomeric to the 200-kb region of ABLbetween exons IB and II in which the breaks occur (24). The BCR probe onchromosome 22, PEM12, is an 18-kb phage clone (in EMBL3) that containspart of, and extends centromeric to, the 5.8-kb breakpoint duster regionof the BCR gene in which almost all CML breakpoints occur. FISH wascarried out using a biotin labeled ABL probe, detected with thefluorochrome Texas red, and a digoxigenin labeled BCR probe, detectedwith the green fluorochrome FITC. Hybridization of both probes could beobserved simultaneously using a fluorescence microscope equipped with adouble band pass filter set (Omega Optical).

[0341]FIG. 8 is a schematic representation of the BCR gene on chromosome22, the ABL gene of chromosome 9, and the BCR-ABL fusion gene on thePhiladelphia chromosome, showing the location of CML breakpoints andtheir relation to the probes. Exons of the BCR gene are depicted assolid boxes. The Roman numeral I refers to the first exon of the BCRgene; the arabic numerals 1-5 refer to the exons within the breakpointcluster region, here indicated by the dashed line. The approximatelocation of the 18 kb phage PEM12 probe (the BCR probe) is indicated bythe open horizontal bar. Since the majority of breakpoints in CML occurbetween exons 2 and 4, 15 kb or more of target for PEM12 will remain onthe Philadelphia chromosome. In the classic reciprocal translocation afew kb of target for PEM12 (undetectable fluorescent signal) will befound on the derivative chromosome. The map and exon numbering (not toscale) is adapted from Heisterkamp et al. (ref. 34, supra).

[0342] Exons of the ABL gene are depicted as open vertical bars (not toscale). The Roman numerals Ia and Ib refer to the alternative firstexons, and II to the second exon. Exon II is approximately 25 kbupstream of the end of the 28 kb cosmid c-hu-abl (the ABL probe). AllCML breakpoints occur upstream of exon II, usually between exons Ib andIa, within a region that is approximately 200 kb in length. Thus,c-hu-abl will always be 25 to 200 kb away from the fusion junction. Themap (not to scale) is adapted from Heisterkamp et al. (ref. 35, supra).The BCR-ABL fusion gene is depicted. In CML, PEM12 will always lie atthe junction, and c-hu-abl will be separated from PEM12 by 25 to 225 kb.

[0343] Sample Preparation: CML-4: Peripheral blood was centrifuged for 5min. Ten drops of interface was diluted with PBS, spun down, fixed inmethanol/acetic acid (3:1), and dropped on slides. CML-2, 3, 7: Five to10 drops of marrow diluted with PBS to prevent clotting were fixed inmethanol/acetic acid and dropped on slides. CML-1,4,5,6: Peripheralblood and/or bone marrow was cultured in RPMI 1640 supplemented with 10%fetal calf serum, an antibiotic mixture (gentamycin 500 mg/ml), and 1%L-glutamine for 24h. Cultures were synchronized according to J. J. Yunisand M. E. Chandler Prog. in Clin. Path., 7:267 (1977), and chromosomepreparations followed Gibis and Jackson, Karyogram, 11:91 (1985).

[0344] Hybridization and Detection Protocol. Hybridization followedprocedures described by D. Pinkel et al. (27), Trask et al. (25), and J.B. Lawrence et al (30), with modifications. The BCR probe wasnick-translated (Bethesda Research Laboratories Nick-Translation System)with digoxigenin-11-dUTP (Boehringer Mannheim Biochemicals) with anaverage incorporation of 25%. The ABL probe was similarlynick-translated with biotin-11-dUTP (Enzo Diagnostics).

[0345] 1. Hybridization. Denature target interphase cells and/ormetaphase spreads on glass slides at 72° C. in 70% formamide/2×SSC at pH7 for 2 min. Dehydrate in an ethanol series (70%, 85%, and 100% each for2 min.). Air dry and place at 37° C. (2xSSC is 0.3M NaCl/30 mM sodiumcitrate). Heat 10 ml of hybridization mixture containing 2 ng/ml of eachprobe, 50% formamide/2×SSC; 10% dextran sulphate, and 1 mg/ml humangenomic DNA (sonicated to 200-600 bp) to 70° C. for 5 min. to denaturethe DNA. Incubate for 30 min. at 37° C. Place on the warmed slides,cover with a 20 mm×20 mm coverslip, seal with rubber cement, andincubate overnight in a moist chamber at 37° C. Remove coverslips andwash three times for 20 minutes each in 50% formamide/2×SSC pH 7 at 42°C., twice for 20 minutes each in 2×SSC at 42° C., and finally rinse atroom temperature in 4×SSC.

[0346] 2. Detection of Bound Probes: All incubation steps are performedwith approximately 100 ml of solution at room temperature undercoverslips. The biotinylated ABL probe was detected first, then thedigoxigenin-labeled BCR probe.

[0347] a. Biotinylated ABL Probe: Preblock with 4×SSC/1% bovine serumalbumin (BSA) for 5 min. Apply Texas Red-avidin (Vector LaboratoriesInc., 2 mg/ml in 4×SSC/1% BSA) for 45 min. Wash in 4×SSC once, 4×SSC/1%Triton-X 100 (Sigma) and then again in 4×SSC, 5 min. each. Preblock for5 min. in PNM (PN containing 5% non-fat dry milk and 0.02% sodium azideand centrifuged to remove solids. PN is 0.1 M NaH₂PO₄/0.1M Na₂HPO₄,0.05% NP40, pH 8). Apply biotinylated goat anti-avidin (VectorLaboratories Inc., 5 mg/ml in PNM) for 45 min. Wash twice in PN for 5min. Apply a second layer of Texas Red-avidin (2 mg/ml in PNM) for 45min. Wash twice in PN for 5 min. each.

[0348] b. Digoxigenin-Labeled BCR Probe: Preblock with PNM for 5 min.Apply sheep anti-digoxigenin antibody (obtained from D. Pepper,Boehringer Mannheim Biochemicals, Indianapolis, Ind.; 15.4 mg/ml in PNM)for 45 min. Wash twice in PN for 5 min. each. Preblock with PNM for 5min. Apply rabbit-anti-sheep antibody conjugated with FITC (OrganonTeknika-Cappel, 1:50 in PNM) for 45 min. Wash twice for 5 min. each inPN. If necessary, the signal is amplified by preblocking for 5 min. withPNM and applying sheep anti-rabbit IgG antibody conjugated to FITC(Organon Teknika-Cappel, 1:50 in PNM) for 45 min. Rinse in PN.

[0349] 3. Visualization: The slides are mounted fluorescence antifadesolution [G. D. Johnson and J. G. Nogueria, J. Immunol. Methods, 43:349(1981)) (ref. 31, supra)] containing 1 mg/ml 4′,6-amidino-2-phenylindole(DAPI) as a counterstain, and examined using a FITC/Texas reddouble-band pass filter set (Omega Optical) on a Zeiss Axioskop.

[0350] The method used for BCR-ABL PCR tested herein was that describedin Hegewisch-Becker et al. for CML-3, 4 and 7 (ref. 32, supra), andKohler et al., for CML-5 and 6 (ref. 33, supra).

[0351] Results. ABL and BCR hybridization sites were visible on bothchromatids of chromosomes in most metaphase spreads. The ABL probe boundto metaphase spreads from normal individuals (FIG. 9 A) near thetelomere on 9q while the BCR probe bound at 22q11 (FIG. 9B).Hybridization with the ABL or BCR probe to normal interphase nucleitypically resulted in two tiny fluorescent dots corresponding to thetarget sequence on both chromosome homologues. The spots were apparentlyrandomly distributed in the two dimensional nuclear images and wereusually well separated. A few cells showed two doublet hybridizationsignals probably a result of hybridization to both sister chromatids ofboth homologues in cells which had replicated this region of DNA (i.e.,those in the S- or G2-phase of cell cycle). Dual color FISH of the ABL(red) and BCR (green) probes to normal G1 nuclei yielded two red (ABL)and two green (BCR) hybridization signals distributed randomly aroundthe nucleus.

[0352] The genetic rearrangement of CML brings the DNA sequenceshomologous to the probes together on an abnormal chromosome, usually thePh¹, and together in the interphase nucleus, as illustrated in FIG. 8.The genomic distance between the probe binding sites in the fusion genevaries among CML cases, ranging from 25 to 225 kb, but remains the samein all the cells of a single leukemic clone. Dual color hybridizationwith ABL and BCR probes to interphase CML cells resulted in one red andone green hybridization signal located at random in the nucleus, and onered-green doublet signal in which the separation between the two colorswas less than 1 micron (or one yellow hybridization signal forhybridization in very close proximity, see FIG. 10). The randomlylocated red and green signals are ascribed to hybridization to the ABLand BCR genes on the normal chromosomes, and the red-green doubletsignal to hybridization to the BCR-ABL fusion gene. Interphase mappingstudies suggest that DNA sequences separated by less than 250 kb shouldbe separated in interphase nuclei by less than 1 micron (25). As aresult, cells showing red and green hybridization signals separated bygreater than 1 micron were scored as normal since this is consistentwith the hybridization sites being on different chromosomes. However,due to statistical considerations, some normal cells will have red andgreen dots close enough together to be scored as abnormal. In these twodimensional nuclear analyses, 9 out of 750 normal nuclei had red andgreen hybridization signals less than 1 micron of each other. Thus,approximately 1% of normal cells were classified as abnormal.

[0353] Table 3 shows the hybridization results for 7 samples from 6 CMLcases along with conventional karyotypes, and other diagnostic results(PCR and Southern blot data ). All six cases, including 3 that werefound to be Ph¹ negative by banding analysis (CML-5,-6 and -7), showedred-green hybridization signals separated by less than 1 micron ingreater than 50% of nuclei examined. In most, the fusion event wasvisible in almost every cell. One case (CML-7) showed fusion signals inalmost every cell even though PCR analysis failed to detect the presenceof a fusion gene and banding analysis did not reveal a Philadelphiachromosome. TABLE 3 Summary of cytogenetic, fluorescence in situhybridization and other analyses of BCR-ABL rearrangements in 6 CMLcases Fluorescence in situ hybridization Sample Cytogenetics MetaphaseInterphase nuclei Other information CML-1^(a) 46XX, t(9; 22)(q34; q11)Hybridization to telomere  80% showed red-green fusion of smallacrocentric  2% showed red-green doublets  18% not interpretableCML-2^(d) 46XY, t(9; 22)(q34; q11) Not available  60% showed red-greenfusion Hybridization efficiency was low CML-3^(d,e) 46XY, t(9; 22)(q34;q11) Not available  75% showed red-green fusion BCR-ABL fusion positive 25% appeared normal by PCR CML-4^(d,e) 46XY, t(9; 22)(q34; q11) Notavailable 100% showed red-green fusion BCR-ABL fusion positive by PCRCML-5^(c) 47XY, +8, del(22)(q11) 47 chromosomes. Red- 100% showedred-green fusion BCR-ABL fusion positive green fusion at telomere by PCRof small acrocentric CML-6^(a) 46XY ins(22; 9)q11; q34; q?) Red-greenfusion 100% showed red-green fusion BCR-ABL fusion positive interstitialon small by PCR acrocentric CML-7^(b) 46XYt(5; 9)q(?; q?) Not available100% showed red-green fusion BCR-ABL fusion negative in two tests by PCRBCR rearrangment detected by Southern blot analysis

[0354] Hybridization to metaphase spreads was performed in three cases(CML-1,-5 and 6). All of these showed red and green hybridizationsignals in close proximity on a single acrocentric chromosome. In twocases, scored as t(9:22)(q34;q11) by banding, the red-green pair was inclose proximity to the telomere of the long arm of a small acrocentricchromosome as expected for the Ph¹ (FIG. 9C). One case (CML6) wassuspected by classical cytogenetics to have an insertion of chromosomalmaterial at 22q11. Dual color hybridization to metaphase spreads fromthis case showed the red-green pair to be centrally located in a smallchromosome (FIG. 9D). That result is consistent with formation of theBCR-ABL fusion gene by an insertion. In one case (CML-1), two pairs ofred-green doublet signals were seen in 3 out 150 (2%) interphase nuclei.That may indicate a double Ph¹ (or double fusion gene) in those cells.Such an event was not detected by standard cytogenetics, which waslimited to analysis of 25 metaphase spreads. The acquisition of anadditional Ph¹ is the most frequent cytogenetic event accompanying blasttransformation, and its cytogenetic detection may herald diseaseacceleration.

[0355] Simultaneous hybridization with ABL and BCR probes to metaphasespreads of the CML derived cell line K-562 showed multiple red-greenhybridization sites along both arms of a single acrocentric chromosome.Hybridization to interphase nuclei showed that the red and green signalswere confined to the same region of the nucleus. That is consistent withtheir being localized on a single chromosome. Twelve to fifteenhybridization pairs were seen in each nucleus indicating correspondingamplification of the BCR-ABL fusion gene (see FIGS. 9E and 9F). Thesefindings are consistent with previous Southern blot data showingamplification of the fusion gene in this cell line (26).

[0356] In summary, analysis of interphase cells for seven CML, and fournormal cell samples using dual color FISH with ABL and BCR probessuggests the utility of this approach for routine diagnosis of CML andclinical monitoring of the disease. Among its very important advantagesare the ability to obtain genetic information from individual interphaseor metaphase cells in less than 24 hours. Thus, it can be applied to allcells of a population, not just to those that fortuitously or throughculture, happen to be in metaphase. Further, the genotypic analysis canbe associated with cell phenotype, as judged by morphology or othermarkers, thereby permitting the study of lineage specificity of cellscarrying the CML genotype as well as assessment of the frequency ofcells carrying the abnormality.

[0357] Random juxtaposition of red and green signals in two dimensionalimages of normal cells, which occurs in about 0.01 of normal cells, setsthe low frequency detection limit. That detection limit may be loweredby more complete quantitative measurement of the separation andintensity of the hybridization signals in each nucleus usingcomputerized image analysis. Such analysis will be particularlyimportant in studying patient populations in which the cells carryingthe BCR-ABL fusion at low frequency (e.g., during remission, after bonemarrow transplantation, during relapse or in model systems).

[0358] This assay also should be advantageous for detection of CML cellsduring therapy when the number of cells available for analysis is lowsince only a few cells are required. Finally, simple counting ofhybridization spots allows for the detection and quantitative analysisof amplification of the BCR-ABL fusion gene as illustrated for the K562cell line (FIG. 9E). Quantitative measurement of fluorescence intensitymay assist with such an analysis.

[0359] The descriptions of the foregoing embodiments of the inventionhave been presented for purpose of illustration and description. Theyare not intended to be exhaustive or to limit the invention to theprecise form disclosed, and obviously many modifications and variationsare possible in light of the above teachings. The embodiments werechosen and described in order to best explain the principles of theinvention and its practical application to thereby enable others skilledin the art to best utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.It is intended that the scope of the invention be defined by the claimsappended hereto.

[0360] All references cited herein are hereby incorporated by reference.

1. A method of staining targeted chromosomal material based upon nucleicacid sequence employing nucleic acid probes wherein said targetedchromosomal material is in the vicinity of a suspected geneticrearrangement.
 2. A method according to claim 1 wherein the targetedchromosomal material is one or more metaphase and/or interphasechromosomes, or one or more regions thereof.
 3. A method according toclaim 2 wherein the chromosomal material is human and selected fromchromosomes 1 through 22, X and Y.
 4. A method according to claim 3wherein the chromosomal material is of fetal cells.
 5. A methodaccording to claim 4 wherein the fetal cells have been separated frommaternal blood.
 6. A method according to claim 1 wherein said nucleicacid probes comprise heterogeneous mixtures of labeled nucleic acidfragments, wherein a substantial fraction of the sequences of thelabeled nucleic acid fragments are substantially complementary to siteson chromosomal material that are targeted and are substantially free ofnucleic acid sequences having hybridization capacity to sites onchromosomal material that is not targeted.
 7. A method according toclaim 1 wherein the genetic rearrangement is selected from the groupconsisting of translocations, inversions, insertions, amplifications anddeletions.
 8. A method according to claim 1 wherein the geneticrearrangement is associated with a disease.
 9. A method according toclaim 8 wherein the genetic rearrangement is associated with cancer. 10.A method according to claim 9 wherein the genetic rearrangement isdiagnostic for chronic myelogenous leukemia (CML).
 11. A methodaccording to claim 10 wherein the genetic rearrangement is selected fromthe group consisting of translocations, deletions, amplifications andinsertions.
 12. A method according to claim 11 wherein said nucleic acidprobes are homologous to nucleic acid sequences in the vicinity of thetranslocation breakpoint regions of chromosomal regions 9q34 and 22q11associated with CML.
 13. A method according to claim 12 wherein saidnucleic acid probes produce a staining pattern which is distinctivelyaltered when the BCR-ABL fusion characteristic of CML occurs.
 14. Amethod according to claim 12 wherein signals from said nucleic acidprobes when hybridized to said nucleic acid sequences produce stainingpatterns as represented in FIG. 11, sections b-e, inclusively.
 15. Amethod according to claim 13 wherein the proximity of and/or othercharacteristics of signals of said staining pattern indicate whethersaid BCR-ABL fusion is present.
 16. A method according to claim 15wherein the portion of the probe to the BCR region is labeled/visualizedin one manner and the portion of the probe to the ABL region islabeled/visualized in another manner so that when the BCR-ABL fusion ispresent the proximity of said two labeling/visualization means becomerelatively close in an interphase and/or metaphase chromosomal spread.17. A method according to claim 14 wherein said nucleic probes have acomplexity of from about 50 kilobases (kb) to about 1 megabase (Mb). 18.A method according to claim 17 wherein the complexity is from about 50kb to about 750 kb.
 19. A method accoridng to claim 18 wherein thecomplexity is from about 200 kb to about 400 kb.
 20. A method accordingto claim 9 wherein the genetic rearrangement occurs relatively closelyin a genome to the location of another genetic rearrangement known to beassociated with cancer.
 21. A method according to claim 20 wherein saidnucleic acid probes produce staining patterns which distinguish agenetic rearrangement associated with either chronic myelogenousleukemia (CML) or acute lymphocytic leukemia (ALL) occurs.
 22. Nucleicacid probes that reliably stain targeted chromosomal materials whereinsaid targeted chromosomal materials are in the vicinity of one or moresuspected genetic rearrangements.
 23. Nucleic acid probes according toclaim 22 that are appropriate for in situ hybridization.
 24. Nucleicacid probes according to claim 23 wherein said nucleic acid sequencesare of sufficient complexity to stain reliably each of two or moretarget sites on chromosomal material in a genome.
 25. Nucleic acidprobes according to claim 24 which are substantially free of nucleicacid sequences having hybridization capacity to sites on non-targetedchromosomal material.
 26. Nucleic acid probes according to claim 22wherein said one or more genetic rearrangements is or are selected fromthe group consisting of translocations, inversions, insertions,amplifications and deletions.
 27. Nucleic acid probes according to claim22 wherein said one or more genetic rearrangements is or are associatedwith one or more diseases.
 28. Nucleic acid probes according to claim 27wherein said one or more genetic rearrangements is or are associatedwith cancer.
 29. Nucleic acid probes according to claim 28 wherein saidone or more genetic rearrangements occurs or occur relatively closely ina genome to another genetic rearrangement known to be associated withcancer.
 30. Nucleic acid probes according to claim 29 wherein saidgenetic rearrangements are associated with either CML and/or ALL. 31.Nucleic acid probes according to claim 29 wherein said probes produce astaining pattern which distinguish genetic rearrangements associatedwith either CML or ALL occurs.
 32. High complexity nucleic acid probesfor the detection of genetic rearrangements.
 33. High complexity nucleicacid probes according to claim 32 wherein the complexity is greater than50,000 bases.
 34. High complexity nucleic acid probes according to claim32 wherein the genetic rearrangements are selected from the groupconsisting of translocations, inversions, insertions, amplifications anddeletions.
 35. High complexity nucleic acid probes according to claim 34wherein said genetic rearrangements are associated with cancer.
 36. Highcomplexity nucleic acid probes according to claim 35 wherein saidgenetic rearrangements are diagnostic for CML.
 37. High complexitynucleic acid probes according to claim 36 wherein said geneticrearrangements are selected from the group consisting of translocations,insertions and amplifications.
 38. High complexity nucleic acid probesaccording to claim 37 which produce a staining pattern which isdistinctively altered when the BCR-ABL fusion characteristic of CMLoccurs.
 39. High complexity nucleic acid probes according to claim 38which have a complexity of from about 50 kb to about 1 megabase. 40.High complexity nucleic acid probes according to claim 39 wherein thecomplexity is from about 50 kb to about 750 kb.
 41. High complexitynucleic acid probes according to claim 40 wherein the complexity is fromabout 200 kb to about 400 kb.
 42. A method of detecting geneticrearrangements comprising the steps of: a. hybridizing the probes ofclaim 32 to targeted chromosomal material in the vicinity of a suspectedgenetic rearrangement; b. observing and/or measuring the proximity ofand/or other characteristics of the signals from said probes; and c.determining from said observations and/or measurements obtained in stepb) whether a genetic rearrangement has occurred.
 43. A method accordingto claim 42 wherein the suspected genetic rearrangement is associatedwith cancer.
 44. A method according to claim 43 wherein the cancer isCML or ALL.
 45. A method according to claim 44 wherein the cancer is CMLand the genetic rearrangement produces a BCR-ABL fusion.
 46. A methodaccording to claim 42 wherein the chromosomal material is either inmetaphase or in interphase.
 47. A method according to claim 46 whereinthe chromosomal material is in metaphase.
 48. A method according toclaim 46 wherein the chromosomal material is in interphase.
 49. A methodaccording to claim 43 wherein the suspected genetic rearrangement isprognostic of cancer.
 50. A method according to claim 49 wherein thecancer is CML.
 51. A method according to claim 50 wherein stainingpatterns produced therefrom are used to distinguish normal and malignantcells for purposes of prognosis and/or determining the effectiveness oftherapy.
 52. A method according to claim 51 wherein said therapeuticregimens are selected from the group consisting of chemotherapy,radiation, surgery and transplantation.
 53. A method according to claim50 wherein staining patterns produced therefrom are useful in monitoringthe status of a patient whose chromosomal material is so tested on acell to cell basis.
 54. A method of determining the molecular basis ofgenetic disease employing the probes of claim
 32. 55. A method ofdistinguishing between CML and ALL based upon staining patterns producedby the method of claim
 44. 56. A method according to claim 53 whereinthe patient is in remission and staining patterns produced therefrom arepredictive of a recurrence of cancer.
 57. High complexity nucleic acidprobes according to claim 52 wherein the targeted chromosomal materialis human and selected from DNA of chromosomes 1 through 22, X and Y. 58.High complexity nucleic acid probes according to claim 32 wherein theprobe nucleic acid sequences are propagated in a cell line and/or in oneor more vectors.
 59. High complexity nucleic acid probes according toclaim 58 wherein said cell line is a hybrid cell line and said one ormore vectors is or are selected from the group consisting of yeastartificial chromosomes, plasmids, bacteriophages and cosmids.
 60. Amethod of staining targeted chromosomal material in the vicinity of asuspected genetic rearrangement with high complexity nucleic acid probesaccording to claim 32 wherein the probe nucleic acid sequences prior tohybridization to the targeted chromosomal material are broken intofragments of from about 200 bases to about 2000 bases.
 61. A methodaccording to claim 60 wherein the size of the fragments are about 1 kb.62. A method according to claim 61 wherein the size of the fragments isfrom about 800 bases to about 1000 bases and, wherein the hybridizationis performed at a temperature of about 30 degrees C. to about 45 degreesC., and wherein the subsequent washing steps are performed at atemperature of from about 40 degrees C. to about 50 degrees C.
 63. Amethod according to claim 62 wherein the hybridization is performed at atemperature of from about 35 degrees C. to about 40 degrees C.
 64. Amethod according to claim 63 wherein the hybridization is performed at atemperature of about 37 degrees C., and the subsequent washing steps areperformed at a temperature of about 45 degrees C.
 65. A method accordingto claim 61 wherein the labeled fragments are detected afterhybridization by flow cytometry.
 66. A method according to claim 60wherein detection is by microscopy.
 67. A method according to claim 66wherein the microscopy is automated.
 68. A method according to claim 65wherein light scattering is used.
 69. High complexity probes accordingto claim 32 wherein the targeted chromosomal material of said probes ischromosomal material of fetal cells.
 70. High complexity probesaccording to claim 69 wherein said fetal cells have been separated frommaternal blood.
 71. High complexity probes according to claim 32 whereinthe targeted chromosomal material is in interphase and/or metaphase. 72.Chromosome-specific staining reagent comprising a heterogeneous mixtureof labeled nucleic acid fragments, wherein the labeled nucleic acidfragments are complementary to sites on targeted chromosomal material inthe vicinity of suspected genetic rearrangements and are substantiallyfree of nucleic acid sequences having hybridization capacity to sites onnon-targeted chromosomal material.
 73. The chromosome-specific stainingreagent of claim 72 wherein said labeled nucleic acid fragments aresingle-stranded.
 74. The chromosome-specific staining reagent of claim73 wherein said nucleic acid fragments are labeled with radioactive,enzymatic, immunoreactive, fluorochromes and/or affinity detectablereagents.
 75. The chromosome-specific staining reagent of claim 74wherein said fragments are biotinylated, modified withN-acetoxy-N-2-acetylaminofluorene, modified with fluoresceinisothiocyanate, modified with mercury/TNP ligand, sulfonated,digoxigenenated, or contain T-T dimers.
 76. A chromosome-specificstaining reagent that provides staining patterns indicative of a geneticrearrangement produced by the process of: isolating chromosome-specificDNA; amplifying pieces of the isolated chromosome-specific DNA;disabling the hybridization capacity of and/or removing sharedrepetitive sequences contained in the amplified pieces of the isolatedDNA to form a collection of nucleic acid fragments which hybridizepredominantly to targeted chromosomal DNA in the vicinity of a suspectedgenetic rearrangement; and labeling the nucleic acid fragments of thecollection to form a heterogeneous mixture of nucleic acid fragments.77. A chromosome-specific staining reagent according to claim 76 whereinsaid step of amplifying said pieces of isolated DNA is performed bycloning.
 78. A chromosome-specific staining reagent according to claim76 wherein said step of amplifying said pieces of isolated DNA isperformed by using the polymerase chain reaction (PCR).
 79. Thechromosome-specific staining reagent of claim 76 wherein said step ofremoving said shared repetitive sequences comprises selecting amplifiedpieces of said isolated DNA which pieces are substantially free ofnucleic acid sequences which are complementary to non-targetedchromosomal material.
 80. The chromosome-specific staining reagent ofclaim 79 wherein said selection of said amplified pieces comprises theuse of Southern hybridization.
 81. The chromosome-specific stainingreagent of claim 80 wherein said selection of said amplified piecescomprises screening clones for the presence of repetitive sequences byhybridization with genomic DNA.
 82. The chromosome-specific stainingreagent of claim 81 wherein said clones are plasmid clones.
 83. Thechromosome-specific staining reagent of claim 82 wherein said selectionof said amplified pieces comprises screening said clones forhybridization to the targeted chromosomal material, and removing theclones which do not so hybridize.
 84. The chromosome-specific stainingreagent of claim 76 wherein said step of disabling said hybridizationcapacity comprises hybridizing said amplified pieces of said isolatedDNA with unlabeled nucleic acid sequences.
 85. The chromosome-specificstaining reagent of claim 84 wherein said step of disabling saidhybridization capacity comprises the addition of unlabeled blockingnucleic acid to the labeled nucleic acid probe prior to and/or duringhybridization to the targeted chromosomal material.
 86. Thechromosome-specific staining reagent of claim 85 wherein the unlabeledblocking DNA is genomic.
 87. The chromosome-specific staining reagent ofclaim 86 wherein the blocking nucleic acid is a high-copy fraction ofgenomic DNA.
 88. The chromosome-specific staining reagent of claim 85wherein the unlabeled blocking DNA is from a selection of clonescontaining the highest copy sequences from a genome and/or additionalclones as required to produce useful contrast.
 89. Thechromosome-specific staining reagent of claim 76 wherein said step ofdisabling said hybridization capacity of said shared repetitivesequences comprises self-reassociating the high complexity probe. 90.The chromosome-specific staining reagent of claim 76 wherein said stepof removing the shared repetitive sequences comprises the use ofhydroxyapatite chromatography.
 91. The chromosome-specific stainingreagent of claim 76 wherein said step of removing the shared repetitivesequences comprises reacting the amplified pieces of the isolated DNAwith immobilized, single-stranded nucleic acid sequences which arecomplementary to said shared repetitive sequences.
 92. A method ofstaining targeted chromosomal material with a high complexity nucleicacid probes according to claim 32 to produce staining patternsindicative of genetic rearrangements wherein unlabeled high copyrepetitive nucleic acid sequences or genomic DNA are hybridized to thetargeted chromosomal material prior to or during the hybridization withthe high complexity nucleic acid probe.
 93. A method of stainingtargeted chromosomal material with high complexity nucleic acid probesaccording to claim 32 to produce staining patterns indicative of geneticrearrangements comprising the steps of: providing a heterogeneousmixture of labeled nucleic acid fragements, wherein substantialproportions of the labeled nucleic acid fragments in the heterogeneousmixture have base sequences substantially complementary to the targetedchromosomal material which is in the vicinity of a suspected geneticrearrangement; reacting the heterogeneous mixture with the targetedchromosomal DNA by in situ hybridization; and observing and/or measuringthe proximity of and/or other characteristics of signals of saidstaining patterns to determine whether a genetic rearrangement hasoccurred.
 94. High complexity nucleic acid probes according to claim 32which are substantially free of shared repetitive sequences produced bya process employing a polymerase chain reaction (PCR) procedure. 95.High complexity nucleic acid probes according to claim 94 wherein duringsaid PCR process, sequences which are complementary to said sharedrepetitive sequences, and which have extended non-complementary ends orwhich are terminated in nucleotides which do not permit extension by apolymerase, are hybridized to said shared repetitive sequences toinhibit amplification of such sequences.
 96. A method of stainingtargeted chromosomal material in the vicinity of a suspected geneticrearrangement with high complexity nucleic acid probes of claim 32wherein the probes are not directly labeled and detection of the probesbound to the targeted chromosomal material is by means other than directlabeling.
 97. A method of staining targeted chromosomal materialaccording to claim 96 wherein the means of detecting the probes bound tothe targeted chromosomal material comprise the use of anti-RNA/DNAduplex antibodies and/or anti-thymidine dimer antibodies.
 98. Highcomplexity nucleic acid probes according to claim 32 for detection ofspecific genetically based diseases.
 99. High complexity nucleic acidprobes according to claim 32 for detection of genetic rearrangementsinduced by exposure to clastogenic agents.
 100. High complexity nucleicacid probes according to claim 99 wherein the probes have been optimizedfor rapid detection of structural chromosome aberrations.
 101. Highcomplexity nucleic acid probes according to claim 32 wherein saidgenetic rearrangements are indicative of cytogenetic abnormalities intumor cells.
 102. Test kits comprising the probes of claim
 32. 103. Testkits comprising the probes of claim
 38. 104. High complexity nucleicacid probes according to claim 32 for use in biological dosimetry. 105.A method of distinguishing between suspected genetic rearrangements thatoccur in relatively close proximity in a genome comprising in situhybridization with nucleic acid probes which comprise sequences whichare substantially homologous to nucleic acid sequences in the vicinityof said suspected genetic rearrangements.
 106. A method according toclaim 105 wherein said suspected genetic rearrangements are associatedwith a disease.
 107. A method according to claim 106 wherein the diseaseis cancer.
 108. A method according to claim 107 wherein said suspectedgenetic rearrangements are associated with CML and ALL.
 109. A method ofdetecting a contiguous gene syndrome comprising the in situhybridization of nucleic acid probes that comprise sequences which aresubstantially homologous to nucleic acid sequences characteristic of oneor more components of said contiguous gene syndrome.
 110. A methodaccording to claim 109 wherein said contiguous gene syndrome is Downsyndrome.
 111. Nucleic acid probes, according to claim 32, comprisingnucleic acid sequences that are substantially homologous to nucleic acidsequences in chromosomal regions that flank and/or extend partially orfully across breakpoints associated with cytogenetically similar butgenetically different diseases.
 112. A method of distinguishingcytogenetically similar but genetically different diseases comprisingthe in situ hybridization of the probes of claim 111 to metaphase and/orinterphase chromosomal spreads.
 113. The method of claim 112 wherein thechromosomal spreads are human.
 114. Nucleic acid probes according toclaim 111 wherein the diseases are cancer.
 115. Nucleic acid probesaccording to claim 114 wherein the diseases are CML and ALL.
 116. Achromosome-specific staining reagent according to claim 72 which isdisease-specific.
 117. A chromosome-specific staining reagent accordingto claim 116 which is tumor-specific.
 118. A chromosome-specificstaining reagent according to claim 17 which is specific for the BCR-ABLfusion characteristic of CML.
 119. A chromosome-specific staining regentaccording to claim 76 which is disease specific.
 120. High complexitynucleic acid probes according to claim 32 comprising nucleic acidsequences substantially homologous to multiple loci in a genome. 121.High complexity nucleic acid probes according to claim 120 wherein saidsequences are associated with regions of the genome in which geneticrearrangements are known to occur.
 122. High complexity nucleic acidprobes according to claim 121 wherein the genome is human.
 123. A methodof simultaneously detecting the genetic rearrangements of multiple lociin a genome comprising in situ hybridization with the probes of claim120.
 124. A method of searching for a genetic rearrangement in achromosomal region of a genome indicated by conventional bandinganalysis to contain an abnormality comprising in situ hybridization ofnucleic acid probes according to claim
 32. 125. A method according toclaim 53 wherein computer assisted microscopic analysis is used tosearch for any residual disease in said patient.
 126. High complexitynucleic acid probes according to claim 32 wherein the probes have beenoptimized for rapid, efficient, automated detection of aberrations.